Methods of treating disease with random copolymers

ABSTRACT

The invention relates to novel methods and kits for treating or preventing disease through the administration of random copolymers comprising amino acids tyrosine (Y), phenylalanine (F), alanine (A), and lysine (K). The invention also relates to the treatment of autoimmune diseases, such as multiple sclerosis, and to the administration of random copolymers in treatment regimen comprising formulations that are administered at intervals greater than 24 hours, or to sustained release formulations which administer the copolymer over a period greater than 24 hours. The invention further relates to methods for conducting a pharmaceutical business comprising manufacturing, licensing, or distributing kits containing or relating to the formulations or dosing regimens of random copolymer described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US05/016340 filed May9, 2005 and PCT/US05/016344 filed May 9, 2005, which applications claimpriority to U.S. Provisional Application Ser. No. 60/569,292 filed May7, 2004, and to U.S. Provisional Application Ser. No. 60/663,333 filedMar. 18, 2005, the entire contents of all of which is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

An autoimmune disease results from an inappropriate immune responsedirected against a self antigen (an autoantigen), which is a deviationfrom the normal state of self-tolerance. Self-tolerance arises when theproduction of T cells and B cells capable of reacting againstautoantigens has been prevented by events that occur in the earlydevelopment of the immune system. The cell surface proteins that play acentral role in regulation of immune responses through their ability tobind and present processed peptides to T cells are the majorhistocompatibility complex (MHC) molecules (Rothbard, J. B., et al.,1991, Annu. Rev. Immunol. 9:527). Autoimmune diseases include rheumatoidarthritis (RA), multiple sclerosis (MS), human type I orinsulin-dependent diabetes mellitus (IDDM), autoimmune uveitis, primarybiliary cirrhosis (PBC) and celiac disease.

One target for inhibition of an autoimmune response is the set oflymphocyte surface protein MHC molecules, particularly a protein encodedby an MHC class II gene, for example, HLA-DR, -DQ and -DP. Each of theMHC genes is found in a large number of alternative or allelic formswithin a mammalian population. The genomes of subjects affected withcertain autoimmune diseases, for example MS and RA, are more likely tocarry one or more characteristic MHC class II alleles, to which thatdisease is linked.

A number of therapeutic agents have been developed to treat autoimmunediseases, including general anti-inflammatory drugs such as COX-2inhibitors, i.e., agents that can prevent formation of low molecularweight inflammatory compounds by inhibiting a cyclooxygenase; agentsthat can function by inhibiting a protein mediator of inflammation, forexample, by sequestering the inflammatory protein tumor necrosis factor(TNF) with an anti-TNF specific monoclonal antibody or antibodyfragment, or with a soluble form of the TNF receptor; and agents thattarget a protein on the surface of a T cell and generally preventinteraction with an antigen presenting cell (APC) by inhibiting the CD4receptor or the cell adhesion receptor ICAM-1. However, compositionshaving natural folded proteins as therapeutic agents can encounterproblems in production, formulation, storage, and delivery. Several ofthese problems necessitate delivery to the patient in a hospitalsetting.

An agent that interacts and binds relatively nonspecifically to severalMHC class II molecules is Copolymer 1 (Cop 1), a synthetic amino acidheteropolymer that was shown to be capable of suppressing experimentalallergic encephalomyelitis (EAE; Sela, M. et al., 1990, Bull. Inst.Pasteur (Paris)), which can be induced in the mouse and is a model forMS. Copolymer 1, which is poly(Y,E,A,K) also known as glatiramer acetateor “YEAK” using the one letter amino acid code (see infra; Y representstyrosine, E glutamic acid, A alanine, and K lysine), has been used totreat relapsing forms of MS but does not suppress the disease entirely(Bornstein, M. B., et al., 1987, N. Engl. J. Med. 317:408; Johnson, K.P. et al., 1995, Neurology 45:1268).

Although random copolymers may be effective for the treatment ofautoimmune diseases (Simpson, D. et al., 2003, BioDrugs 17(3):207-10),their repeated administration may cause undesired side effects.Accordingly, there is a need for improved methods for the treatment ofautoimmune diseases with random copolymers which result in fewer sideeffects.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods and kits for the treatment or preventionof disease in a subject, preferably in a human. One aspect of theinvention provides methods of treating or preventing a disease, themethod comprising administering to said subject a dosing regimen of aneffective amount of a random copolymer for the amelioration of a diseasetreatable with the random copolymer, said effective amount delivered tosaid subject at time intervals greater than 24 hours, 36 hours, or morepreferably greater than 48 hours. A related aspect of the inventionprovides a method for the treatment of a subject in need thereof,comprising administering to said subject a dosing regimen of aneffective amount of a random copolymer for the amelioration of a diseasetreatable with the random copolymer, said effective amount delivered tothe subject using a sustained-release formulation which administers therandom copolymer over a period of at least 2 days, at least 4 days, orat least 6 days, wherein the effective amount is an amount that iseffective if delivered daily. In some embodiments, the disease of themethods of the present invention is mediated by T-cells, and inparticular T_(H)1 cells or cells with T_(H)1 immune posture, or is adisease which is exacerbated by an excess of inflammatory cytokines. Insome embodiments, the disease is an autoimmune disease, such as multiplesclerosis. In some preferred embodiments, the random copolymer comprisestyrosine (Y), phenylalanine (F), alanine (A) and lysine (K) (YFAKcopolymer). In other embodiments, the random copolymer is Copolymer 1(YEAK). In particular, the method of present invention further comprisesadministering to said subject an anti-lymphocyte therapies. In oneembodiment, the anti-lymphocyte therapy comprises administering an agentselected from the group consisting of a polyclonal antibody or amonoclonal antibody. In certain embodiments, the polyclonal antibody isantithymocyte gamma globulin (ATGAM). In other embodiment, the antibodyis a monoclonal antibody selected from the group consisting ofalemtuzumab (Campath®), muromonab (OKT®3), daclizumab, and basiliximab.In another embodiment, the method of the invention further comprisesadministering to said subject an anti B-cell therapy. In one embodiment,the anti-B-cell therapy comprises administering anti CD-20 antibody. Oneaspect of the invention is a method treating a disease treatable byadministering a random copolymer composition comprising administering toa subject in need thereof a dosing regimen of an effective amount of arandom copolymer composition for the amelioration of said disease,wherein the disease is selected from the group consisting of allergies,asthma, atopic dermatitis, and neuroprotection.

The invention is not limited to any particular random copolymer or modeof administration.

The invention also provides kits for the treatment of disease. Oneaspect of the invention provides a kit for the treatment of anautoimmune disease comprising (i) a composition comprising a randomcopolymer and (ii) instructions for administering the composition to asubject at time intervals of at least 24 hours, or more preferably 36 or48 hours or longer. In preferred embodiments, the composition isformulated for subcutaneous injection, the random copolymer is YFAK orCopolymer 1, and the disease is an autoimmune disease, such as multiplesclerosis, particularly relapsing-remitting multiple sclerosis.

The invention further provides agents for the manufacture of medicamentsto treat diseases. Any methods disclosed herein for treating orpreventing a disease by administering a random copolymer to a subjectmay be applied to the use of the random copolymer in the manufacture ofa medicament to treat that disease. Accordingly, one aspect of theinvention provides the use of a random copolymer for the treatment of adisease in a subject, wherein the random copolymer is formulated to beadministered to the subject at intervals greater than 24 hours, 36hours, and more preferably of at least 48 hours. In preferredembodiments, the random copolymer is Copolymer 1 (YEAK), and the diseaseis an autoimmune disease, such as multiple sclerosis, particularlyrelapsing-remitting multiple sclerosis.

The invention further provides methods of conducting a pharmaceuticalbusiness.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of copolymer administration on the diseaseprogression of EAE.

FIG. 2. shows the survival rate of mice with EAE when administered withrandom copolymers.

FIG. 3 shows IgG antibody production against copolymers administered atdaily or weekly doses.

FIG. 4 shows IgG1 antibody production against copolymers administered atdaily or weekly doses.

FIG. 5 shows IgG2b antibody production against copolymers administeredat daily or weekly doses.

FIG. 6 shows the changes in antibody titer against copolymers during thetime course of a treatment.

FIG. 7 shows the IgG1 antibody production against PLP peptide in miceadministered with random copolymers.

FIG. 8 shows the IgG2b antibody production against PLP peptide in miceadministered with random copolymers.

FIG. 9 shows the ratio of IL-13 over IFN γ in mice administered withrandom copolymers.

FIG. 10 shows the bias for induction of TH2 related cytokines comparedto TH1 related cytokines in mice administered with random copolymers.

FIG. 11 shows the ability of Co-14 to generate specific effects upon Tand B cells.

FIG. 12 shows the dose dependent antibody response (IgG) to Co-14.

FIG. 13A-C shows opposing TH1/TH2 ratios which are dose, regimen, andadministration dependent. The recall response shown is representative ofboth of the two TH1/TH2 conditions.

FIG. 14 shows the ability of Co-14 to mediate recall responses to aMyasthenia Gravis-associated acetyl choline receptor peptide.

FIG. 15 shows the ability of the proper ratio of YFAK within the Co-14RSP to generate a recall response in a NOD mouse.

FIG. 16 shows the differential induction of peripheral and centraltolerance.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The invention broadly relates to the treatment and prophylaxis ofdiseases by the administration of random copolymers, to the use of therandom copolymers in the manufacture of medicaments to treat disease,and to kits comprising both random copolymers and instructions. Theinvention also relates to the treatment of autoimmune diseases and tolong-lasting random copolymer formulations for the treatment of disease.

One aspect of the invention provides a method for the treatment of asubject comprising administering to said subject a dosing regimen of aneffective amount of a random copolymer for the amelioration of a diseasetreatable with the random copolymer, said effective amount delivered tosaid subject at time intervals greater than 36 hours. A related aspectof the invention provides a method for the treatment of a subjectcomprising administering to said subject a dosing regimen of aneffective amount of at least one random copolymer for the ameliorationof a disease treatable with the random copolymer, said effective amountof at least one copolymer being delivered to said subject at timeintervals greater than 24 hours, and in particular greater than 48hours. In one embodiment, the effective amount of the random copolymerthat is administered at intervals greater than 24 hours is an amountthat is effective when administered daily. In a related embodiment, theeffective amount that is administered at intervals greater than 24 hoursis an amount that would be effective if administered daily. In yetanother related embodiment, the effective amount that is administered atintervals greater than 24 hours is an amount that is known to beeffective if administered daily. In an embodiment of this invention, theeffective amount consists of between 10 mg and 30 mg, or between 15 mgand 25 mg. In other embodiments, the effective amount is about 20 mg. Inanother embodiment, the effective amount is less than 20 mg. In specificembodiments, the effective amount is “x” mg, wherein “x” is any integerbetween 1 and 20.

In one embodiment of the methods provided herein, the subject isafflicted with a disease treatable with the random copolymer. In oneembodiment, the disease is mediated by T-cells, and in particular T_(H)1cells or cells with T_(H)1 immune posture, or is a disease which isexacerbated by an excess of inflammatory cytokines. In anotherembodiment, the subject is afflicted with at least one autoimmunedisease. In one embodiments, the subject is afflicted with at least onedisease selected from the group consisting of multiple sclerosis, type-Idiabetes, Hashimoto's thyroiditis, Crohn's disease, rheumatoidarthritis, systemic lupus erythematosus (SLE), gastritis, autoimmunehepatitis, hemolytic anemia, autoimmune hemophilia, autoimmunelymphoproliferative syndrome (ALPS), autoimmune uveoretinitis,glomerulonephritis, Guillain-Barré syndrome, psoriasis, myastheniagravis, autoimmune encephalomyelitis, Goodpasture's syndrome, Grave'sdisease, paraneoplastic pemphigus, autoimmune thrombocytopenic purpura,scleroderma with anti-collagen antibodies, mixed connective tissuedisease, pernicious anemia, polymyositis, idiopathic Addison's disease,autoimmune-associated infertility, glomerulonephritis, bullouspemphigoid, Sjogren's syndrome, idiopathic myxedema and colitis. Inpreferred embodiments, the disease is multiple sclerosis orrelapsing-remitting multiple sclerosis. In additional embodiments of themethods provided herein, the disease is host-versus-graft disease (HVGD)or graft-versus-host disease (GVHD) or both. In preferred embodiments ofthe methods described herein, the subject is a mammal, or morepreferably a human.

In one embodiment of the methods described herein, the dosing regimencomprises intravenous, subcutaneous, intramuscular, intradermal,intraperitoneal, intradermal or oral administration. The randomcopolymer may also be administered via devices designed to deliver therandom copolymer continuously, such as a transdermal patch or pump orimplant. For example, a transdermal patch may be used to administer therandom copolymer over a span of 12 hours every 48 hours or longer, or apump may be used to administer the copolymer over a period of two daysevery four or more days. In a related aspect, the copolymer isadministered in a sustained release formulation.

The invention also provides a method for the treatment of a subject inneed thereof comprising administering to said subject a dosing regimenof an effective amount of a random copolymer for the amelioration of adisease treatable with the random copolymer, said effective amountdelivered to the subject using a sustained-release formulation whichadministers the random copolymer over a period of at least 2 days, atleast 4 days, or at least 6 days, wherein the effective amount is anamount that is effective if delivered daily. In preferred embodiments,the sustained release formulation administers the copolymer over aperiod of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days. Inanother embodiment, the total dosage delivered daily by the sustainedrelease formulation is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%,10% or 5% of a daily dosage known to be effective in the treatment ofthe disease. In a specific embodiment, the sustained release formulationadministers 25% or less, per day, of a dosage of a random copolymerwhich is known to be effective in treating the disease when administereddaily. As an illustrative example, if Copolymer 1 (YEAK) is known to beeffective in the treatment of relapsing-remitting multiple sclerosiswhen administered daily in dosages of 20 mg, such as by one dailysubcutaneous injection of 20 mg, the invention provides sustainedrelease formulations of Copolymer 1 which results in a dailyadministration of copolymer of less than 20 mg, and in particular lessthan about 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg or 1 mgof Copolymer 1.

In some embodiments of the methods described herein, the methods furthercomprise administering an additional therapeutically active agent to thesubject, such as an anti-inflammatory agent. In preferred embodiments,the agent is useful in treating the disease. In another preferredembodiment, the agent synergizes with the random copolymer to treat thedisease.

In some embodiments of the methods described herein, the dosing regimencomprises administering the random copolymer to the subject multipletimes, with a time interval between each administration. In preferredembodiments, the time interval between each administration is at least36, 48, 72, 96, 120, or 144 hours. In another preferred embodiment, thetime interval between each administration is between 36 hours and 14days, or at least 7 days. In a related embodiment, at least one of thetime intervals between administrations is at least 36, 48, 72, 96, 120,or 144 hours, at least 7 days, or between 36 hours and 14 days. Inanother related embodiment, at least 10%, 20%, 30%, 40% or morepreferably 50% of the time intervals between administrations are atleast 36, 48, 72, 96, 120, or 144 hours, at least 7 days, or between 36hours and 14 days. In yet another related embodiment, the average timeinterval between administrations is at least 36, 48, 72, 96, 120, or 144hours, at least 7 days, or between 36 hours and 14 days.

In some embodiments of the methods described herein, the effectiveamount of the random copolymer is between 0.02 mg per dose and 2000 mgper dose, or more preferably between 2 mg per dose and 200 mg per dose.

In some embodiments of the methods described herein, the randomcopolymer is selected from the group consisting of Copolymer 1 (YEAK),YFAK, VYAK, VWAK, VEAK and FEAK. In a preferred embodiment, the randomcopolymer is Copolymer 1. In another preferred embodiment, the randomcopolymer is YFAK. In another embodiment, the random copolymer is aterpolymer, such as one selected from the group consisting of YAK, YEK,KEA and YEA. In yet another embodiment, the random copolymer has betweenone and 10 anchor residues.

The invention also provides kits for the treatment of disease. Oneaspect of the invention provides a kit for the treatment of anautoimmune disease comprising (i) a composition comprising a randomcopolymer and (ii) instructions for administering the composition to asubject at time intervals of at least 36 hours. In a preferredembodiment, the random copolymer in the kit is Copolymer 1. In anotherpreferred embodiments, the random copolymer in the kit is YFAK. In someembodiments, the random copolymer in the kit is formulated foradministration every about 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84,90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174,180, 186, 192, 198, 204, 210, 216, 222, 228, 234, or 240 hours. In someembodiments, the instructions of the kit indicate that the randompolymer is to be administered to the subject at time intervals of atleast 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114,120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198,204, 210, 216, 222, 228, 234, or 240 hours.

In some embodiments of the kits provided by the invention, thecomposition is formulated as a sustained release formulation. Inspecific embodiments, the sustained release formulation delivers a totaldosage that would be effective in treating the disease if said totaldosage were administered daily. In other embodiments, the total dosageis about 20 mg, less than 20 mg, or x mg, wherein x is any integerbetween 1 and 20.

In another embodiment of the kits provided by the invention, the kitcomprises instructions for administering the composition to a subject inneed thereof at time intervals of at least 24, 36, 48, 72, 96, 120 or144 hours or longer, at a dosage of about 20 mg per administration,while in other embodiments the dosage is less than 20 mg, such as x mg,wherein x is any integer between 1 and 20. In a related embodiment, thekit comprises instructions for administering the composition to asubject in need thereof at time intervals of at least 24 hours at adosage that is effective in treating the disease if it were to beadministered daily. In another related embodiment, the kit comprisesinstructions for administering the composition to a subject in needthereof at time intervals of at least 24 hours at a dosage that iseffective in treating the disease when administered daily.

In some embodiments, the disease for which the kit is directed ismediated by T-cells, and in particular T_(H)1 cells, or the disease isone which is exacerbated by an excess of inflammatory cytokines. Inanother embodiments, the disease is an autoimmune disease for which thekit provides treatment is selected from the group consisting of multiplesclerosis, type-I diabetes, Hashimoto's thyroiditis, Crohn's disease,rheumatoid arthritis, systemic lupus erythematosus (SLE), gastritis,autoimmune hepatitis, hemolytic anemia, autoimmune hemophilia,autoimmune lymphoproliferative syndrome (ALPS), autoimmuneuveoretinitis, glomerulonephritis, Guillain-Barré syndrome, psoriasis,myasthenia gravis, autoimmune encephalomyelitis, Goodpasture's syndrome,Grave's disease, paraneoplastic pemphigus, autoimmune thrombocytopenicpurpura, scleroderma with anti-collagen antibodies, mixed connectivetissue disease, pernicious anemia, polymyositis, idiopathic Addison'sdisease, autoimmune-associated infertility, bullous pemphigoid,Sjogren's syndrome, idiopathic myxedema and colitis. In specificembodiments, the disease is multiple sclerosis, diabetes or arthritis.In a preferred embodiment, the disease is relapsing-remitting multiplesclerosis. The kit may also comprise packaging and a means ofadministrating the copolymer, such as a hypodermic syringe, needles,measuring devices such as a spoon or graduated container, an inhaler ora pump. The instructions on the kit may also contain instructions forhome use.

The invention further provides agents for the manufacture of medicamentsto treat diseases. Any methods disclosed herein for treating orpreventing a disease by administering a random copolymer to a subjectmay be applied to the use of the random copolymer in the manufacture ofa medicament to treat that disease. Accordingly, one aspect of theinvention provides the use of a random copolymer for the treatment of adisease in a subject, wherein the random copolymer is formulated to beadministered to the subject at intervals greater than 24 hours, and morepreferably of at least 48 hours. In preferred embodiments, the randomcopolymer is Copolymer 1, and the disease is an autoimmune disease, suchas multiple sclerosis or more particularly relapsing-remitting multiplesclerosis. In other preferred embodiments, the random copolymer is YFAK.

Another aspect of the invention provides for certain methods of doingbusiness. In particular, the invention provides methods of conducting apharmaceutical business wherein the kits and formulations are marketedto healthcare providers or directly to subjects in need of such kits.One aspect provides a method for conducting a pharmaceutical business,comprising marketing to healthcare providers, or to patients in need ofsuch kits, the benefits of using any of the kits described herein in thetreatment of a disease or disorder. A related aspect provides a methodfor conducting a pharmaceutical business, comprising: (a) manufacturingany of the kits described herein; and (b) marketing to healthcareproviders, or to patients in need of such kits, the benefits of usingthe kit in the treatment of a disease or disorder. In some embodiments,the rights to develop and market such formulations or to conduct suchmanufacturing steps may be licensed to a third party for consideration.In some embodiments, the disease is multiple sclerosis, such asrelapse-remitting multiple sclerosis. In another embodiment, the kitscomprise Copolymer 1 or YFAK.

In another embodiment, the marketing to healthcare providers or topatients comprises an indication to administer 50 mg, or more preferably20 mg or less of the random copolymer every 5 to 7 days. In otherembodiments, the marketing comprises an indication to administer therandom copolymer every at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13or 14 days. In another embodiment, the marketing to healthcare providersor to patients comprises an indication to administer 50 mg, or morepreferably 20 mg or less of the random copolymer, every 5 to 7 days. Inyet another embodiment, the marketing comprises an indication of reducedside effects in using the kits or formulations described herein comparedto existing formulations of the same or a different random copolymer. Ina specific embodiment, the existing formulations are administered morefrequently to the patient, or with shorter intervals betweenadministrations, while in another embodiment the existing formulationsresult in a higher average daily dosage than those of the kit that ismarketed. The higher average daily dosage may be, for example, 20, 50,100, 200, or 500% higher than those provided by the kits.

II. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims, are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited” to.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

The term “such as” is used herein to mean, and is used interchangeably,with the phrase “such as but not limited to”.

A “patient” or “subject” to be treated by the method of the inventioncan mean either a human or non-human animal, preferably a mammal.

The term “autoimmune condition” or “autoimmune disease” means a diseasestate caused by an inappropriate immune response that is directed to aself-encoded entity which is known as an autoantigen. The copolymercompounds provided herein can be used to treat symptoms of an autoimmunedisease, a class of disorder which includes Hashimoto's thyroiditis;idiopathic myxedema, a severe hypothyroidism; multiple sclerosis, ademyelinating disease marked by patches or hardened tissue in the brainor the spinal cord; myasthenia gravis which is a disease havingprogressive weakness of muscles caused by autoimmune attack onacetylcholine receptors at neuromuscular junctions; Guillain-Barresyndrome, a polyneuritis; systemic lupus erythematosis; uveitis;autoimmune oophoritis; chronic immune thrombocytopenic purpura; colitis;diabetes; Grave's disease, which is a form of hypothyroidism; psoriasis;pemphigus vulgaris; and rheumatoid arthritis (RA).

The term “demyelinating condition” includes a disease state in which aportion of the myelin sheath, consisting of plasma membrane wrappedaround the elongated portion of the nerve cell, is removed bydegradation. A demyelinating condition can arise post-vaccination,post-anti TNF treatment, post-viral infection, and in MS.

The term “derivative” of an amino acid means a chemically related formof that amino acid having an additional substituent, for example,N-carboxyanhydride group, a γ-benzyl group, an ε-N-trifluoroacetylgroup, or a halide group attached to an atom of the amino acid.

The term “analog” means a chemically related form of that amino acidhaving a different configuration, for example, an isomer, or aD-configuration rather than an L-configuration, or an organic moleculewith the approximate size, charge, and shape of the amino acid, or anamino acid with modification to the atoms that are involved in thepeptide bond, so that the copolymer having the analog residue is moreprotease resistant than an otherwise similar copolymer lacking suchanalog, whether the analog is interior or is located at a terminus ofthe copolymer, compared to the copolymer without the analog.

The phrases “amino acid” and “amino acid copolymer” can include one ormore components which are amino acid derivatives and/or amino acidanalogs as defined herein, the derivative or analog comprising part orthe entirety of the residues for any one or more of the 20 naturallyoccurring amino acids indicated by that composition. For example, in anamino acid copolymer composition having one or more tyrosine residues, aportion of one or more of those residues can be substituted withhomotyrosine. Further, an amino acid copolymer having one or morenon-peptide or peptidomimetic bonds between two adjacent residues isincluded within this definition.

The term “hydrophobic” amino acid means aliphatic amino acids alanine(A, or ala), glycine (G, or gly), isoleucine (I, or ile), leucine (L, orleu), methionine (M, or met), proline (P, or pro), and valine (V, orval), the terms in parentheses being the one letter and three letterstandard code abbreviations for each amino acid, and aromatic aminoacids tryptophan (W, or trp), phenylalanine (F, or phe), and tyrosine(Y, or tyr). These amino acids confer hydrophobicity as a function ofthe length of aliphatic and size of aromatic side chains, when found asresidues within a copolymer or other polypeptide.

The term “charged” amino acid means amino acids aspartic acid (D orasp), glutamic acid (E or glu), arginine (R or arg) and lysine (K orlys), which confer a positive (lys, and arg) or negative (asp, glu)charge at physiological values of pH on an aqueous solution of acopolymer or other amino acid composition containing one or moreresidues of these amino acids. Histidine (H or his) is hydrophobic at pH7, and charged at pH 6.

The terms “disorders” and “diseases” are used inclusively and refer toany deviation from the normal structure or function of any part, organor system of the body (or any combination thereof). A specific diseaseis manifested by characteristic symptoms and signs, includingbiological, chemical and physical changes, and is often associated witha variety of other factors including, but not limited to, demographic,environmental, employment, genetic and medically historical factors.Certain characteristic signs, symptoms, and related factors can bequantitated through a variety of methods to yield important diagnosticinformation.

The term “prophylactic” or “therapeutic” treatment refers toadministration to the subject of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it contributes toprotection of the host against developing the unwanted condition,whereas if administered after manifestation of the unwanted condition,the treatment is therapeutic (i.e., it is intended to diminish,ameliorate or prevent progression of the unwanted condition or sideeffects therefrom).

The term “therapeutic effect” refers to a local or systemic effect inanimals, particularly mammals, and more particularly humans, caused by apharmacologically active substance. The term thus means any substanceintended for use in the diagnosis, cure, mitigation, treatment orprevention of disease or in the enhancement of desirable physical ormental development and conditions in an animal or human. The phrase“therapeutically-effective amount” means that amount of such a substancethat produces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. In certain embodiments,a therapeutically-effective amount of a compound will depend on itstherapeutic index, solubility, and the like. For example, certaincompounds discovered by the methods of the present invention may beadministered in a sufficient amount to produce a reasonable benefit/riskratio applicable to such treatment.

The term “effective amount” refers to the amount of a therapeuticreagent that when administered to a subject by an appropriate dose andregimen produces the desired result.

The term “subject in need of treatment for a disorder” is a subjectdiagnosed with that disorder, likely to develop the disorder, or issuspected of having that disorder.

The term “antibody” as used herein is intended to include wholeantibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includesfragments thereof which are also specifically reactive with avertebrate, e.g., mammalian, protein. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility and/orinteraction with a specific epitope of interest. Thus, the term includessegments of proteolytically-cleaved or recombinantly-prepared portionsof an antibody molecule that are capable of selectively reacting with acertain protein. Non-limiting examples of such proteolytic and/orrecombinant fragments include Fab, F(ab′)2, Fab′, Fv, and single chainantibodies (scFv) containing a V[L] and/or V[H] domain joined by apeptide linker. The scFv's may be covalently or non-covalently linked toform antibodies having two or more binding sites. The term antibody alsoincludes polyclonal, monoclonal, or other purified preparations ofantibodies and recombinant antibodies.

The term “central tolerance” means tolerance for an antigen controlledby the events in the thymus, namely the clonal deletion of T cellsreactive to the antigen in the thymus gland. Partially activated T cellswith high affinity receptors for the antigen undergo negative selectionand clonal deletion in the thymus by Fas-mediated apoptosis, triggeredby coexpression and binding of FasL to Fas on the cell surface. Incontrast, the term “peripheral tolerance” means deletion of T cells byactivation-induced cell death (AICD) and functional silencing (clonalanergy) of T cells without clonal deletion in the spleen. Also, whenlacking the cooperation of helper T cells, B cells are presumably“helpless” to respond to T cell dependent antigens. The modulation ofcentral and peripheral tolerance is regulated by phosphorylation ofp56^(lck) and ZAP-70. The status and the degree of phosphorylation ofkey residues of these proteins result in up or down regulation ofsignaling molecules that influence the peripheral and central tolerance.Inhibition of T cell receptor signaling also plays a role in inducingtolerance.

Other technical terms used herein have their ordinary meaning in the artthat they are used, as exemplified by a variety of technicaldictionaries.

III. Random Copolymers

The composition of a random copolymer of the instant invention comprisesthe characteristics of a compilation of a multiplicity of cross-reactiveT cell epitopes. The composition of a random copolymer of the instantinvention may further comprise the characteristics of altered peptideligands. Multiple functional consequences of the composition of a randomcopolymer of the instant invention exist: one is the potential tofunctionally interact with thousands, preferably hundreds of thousands,more preferably millions, of T cell epitopes via presentation by MHCmolecules, preferably MHC class II molecules, while another is thegeneration of random copolymer specific T cells which may secretesoluble mediators, such as cytokines.

A random copolymer of the instant invention may be given specific aminoacid sequence characteristics such that the selected sub-group of aminoacids preferentially interacts with specific T cell epitopes, some ofwhich may be directly associated with pathogenic disorders. Preferably,a random copolymer of the instant invention may be given specific aminoacid sequence characteristics such that the selected sub-group of aminoacids comprises between two and eight amino acids which preferentiallyinteract with specific T cell epitopes some of which may be directlyassociated with pathogenic disorders which are exacerbated by aberrantproduction of soluble mediators, such as cytokines.

Preferably, a random copolymer of the instant invention may be givenspecific amino acid sequence characteristics such that the selectedsub-group of amino acids comprises between two and eight amino acidswhich by virtue of the amino acids chosen and the ratio of said aminoacids to one another preferentially interact with specific T cellepitopes some of which may be directly associated with pathogenicdisorders which are exacerbated by aberrant production of solublemediators, such as cytokines said pathogenic disorders having linkage tospecific MHC class II alleles such as HLA-DR, or HLA-DQ.

More preferably, a random copolymer of the instant invention comprises apolymer of from two to eight amino acids randomly connected, preferablyvia peptide bonds which preferentially interacts with specific T cellepitopes some of which may be directly associated with pathogenicdisorders which are exacerbated by aberrant production of solublemediators, such as cytokines, said pathogenic disorders having linkageto specific MHC class II alleles such as HLA-DR, or HLA-DQ.

More preferably, a random copolymer of the instant invention comprises apolymer of from three to five amino acids randomly connected, preferablyvia peptide bonds which preferentially interacts with specific T cellepitopes some of which may be directly associated with pathogenicdisorders which are exacerbated by aberrant production of solublemediators, such as cytokines, said autoimmune disorders having linkageto specific MHC class II alleles such as HLA-DR, or HLA-DQ.

The random copolymers of the present invention may comprise a suitablequantity of an amino acid of positive electrical charge, such as lysineor arginine, in combination with an amino acid with a negativeelectrical charge (preferably in a lesser quantity), such as glutamicacid or aspartic acid, optionally in combination with an electricallyneutral amino acid such as alanine or glycine, serving as a filler, andoptionally with an amino acid adapted to confer on the copolymerimmunogenic properties, such as an aromatic amino acid like tyrosine ortryptophan. Such compositions may include any of those disclosed in WO00/005250, the entire contents of which being hereby incorporated hereinby reference.

Copolymers Comprising Four Amino Acids

In one embodiment of the invention, the random copolymer contains fourdifferent amino acids, each from a different one of the followinggroups: (a) lysine and arginine; (b) glutamic acid and aspartic acid;(c) alanine and glycine; (d) tyrosine and tryptophan.

A specific copolymer according to this embodiment of the presentinvention comprises in combination alanine, glutamic acid, lysine, andtyrosine, and has a net overall positive electrical charge. Onepreferred example is YEAK, also referred to as Copolymer 1 (Cop 1) orglatiramer acetate, of average molecular weight about 4,700 to about13,000 daltons. A preferred copolymer has a molecular weight of about2,000 to about 40,000 daltons, or from about 2,000 to about 13,000daltons. Preferred molecular weight ranges and processes for making apreferred form of Copolymer 1 are described in U.S. Pat. No. 5,800,808,the entire contents of which are hereby incorporated in the entirety.Thus, the copolymer may be a polypeptide from about 15 to about 100,preferably from about 40 to about 80, amino acids in length. In apreferred embodiment, the length of Copolymer 1 is between 35 and 75amino acids residues. More preferably, the length of Copolymer 1 isbetween 35 and 65 amino acid residues. In a preferred embodiment thelength of Copolymer 1 is about 50 amino acids. In another preferredembodiment, the length of Copolymer 1 is about 52 amino acids. In apreferred embodiment, Copolymer 1 has an average molar output ratio ofabout 1.0:2.0:6.0:5.0 for Y:E:A:K respectively, synthesized by solidphase chemistry as described below in more detail. The variability inthe output ratios comprises a range of about 10% between the differentamino acids.

In a preferred embodiment of Copolymer 1 of about 52 amino acidresidues, the ratio of alanine composition in amino acid positions 31-52is greater than in amino acid positions 11-30, and the ratio of alaninecomposition in amino acid positions 11-30 is greater than in amino acidpositions 1-10. More specifically, a preferred embodiment of theinvention is a random copolymer of the composition YEAK (L-tyrosine,L-glutamate, L-alanine and L-lysine) in an average molar output ratio ofabout 1.0:2.0:6.0:5.0 respectively, synthesized by solid phasechemistry, wherein the copolymer has a length of 52 amino acids, andwherein residues 1-10 of the copolymer sequence has a molar output ratioof about 1.0:2.0:5.5:5.0, residues 11-30 have a molar output ratio ofabout 1.0:2.0:6.0:5.0, and residues 31-52 have a molar output ratio ofabout 1.0:2.0:6.5:5.0.

For the purpose of the present invention, “Cop 1 or a Cop 1-relatedpeptide or polypeptide” is intended to include any peptide orpolypeptide, including a random copolymer, that cross-reactsfunctionally with myelin basic protein (MBP) and is able to compete withMBP on the MHC class II in the antigen presentation. Copolymer 1 hasbeen approved in several countries for the treatment of multiplesclerosis (MS) under the trade name, COPAXONE™. COPAXONE™ is a trademarkof Teva Pharmaceuticals Ltd., Petah Tikva, Israel. Copolymer 1 bindswith high affinity and in a peptide-specific manner to purifiedMS-associated HLA-DR2 (DRB1*1501) and rheumatoid arthritis(RA)-associated HLA-DR1 (DRBI*0101) or HLA-DR4 (DRB1*0401) molecules.Since Copolymer 1 is a mixture of random polypeptides, it may containdifferent sequences that bind to different HLA proteins; in this caseonly a fraction out of the whole mixture would be an “active component.”Alternatively, the whole mixture may be competent, i.e. all polypeptidesbinding to any HLA-DR molecule.

More preferably, a random copolymer of the instant invention comprises apolymer of the amino acids Copolymer 1 or YFAK randomly connected viapeptide bonds which preferentially interacts with specific T cellepitopes associated with autoimmune disorders which are exacerbated byaberrant production inflammatory cytokines, said autoimmune disordershaving linkage to specific MHC class II alleles such as HLA-DR, orHLA-DQ.

More preferably, a random copolymer of the instant invention comprises apolymer of the amino acids Copolymer 1 or YFAK randomly connected viapeptide bonds which preferentially interacts with specific T cellepitopes associated with rheumatoid arthritis, multiple sclerosis,diabetes, celiac disease, rheumatoid arthritis, steroid sensitivenephrotic syndrome, mesengial IgA nephropathy, narcolepsy, neurologicalmultiple sclerosis, relapsive polychondritis, dermatological disorderssuch as dermatitis herpetiformis, atopic dermatitis, Behcet's disease,pemphigus, psoriasis, primary Sjögren's syndrome, systemic vasculitides,erythematosus, gastrointestinal disorders such as Crohn's disease,respiratory disorders such as Sommer type hypersensitivity pneumonitis,and autoimmune thyroid disease (AITD).

In another aspect of the invention, the random copolymer comprises YFAK,VYAK, VWAK, VEAK and FEAK. In a preferred embodiment, the randomcopolymer consists of amino acid residues YFAK (L-tyrosine,L-phenylalanine, L-alanine and L-lysine) in an molar output ratio ofabout 1.0:1.2:XA:6.0 respectively, wherein XA is greater than 11.0 andless than 30.0, and the variability in the output ratios comprises arange of about 10% between the different amino acids. In anotherpreferred embodiment, the random copolymer consists of amino acidresidues YFAK in a molar output ratio of about 1.0:1.0:XA:6.0respectively, wherein XA is greater than 5.0 and less than 15.0, and thevariability in the output ratios comprises a range of about 10% betweendifferent amino acids. The molar output ratios of YFAK of randomcopolymers of the preferred embodiments are shown in Table I below:

TABLE I Amino Acid Composition Ratios of Random Copolymers Y F A K 1.0:1.2: 11.0 < 30.0: 6.0 1.0: 1.2: 16.0: 6.0 1.0: 1.2: 18.0: 4.0 1.0: 1.2:18.0: 5.0 1.0: 1.2: 18.0: 6.0 1.0: 1.2: 18.0: 7.0 1.0: 1.2: 18.0: 8.01.0: 1.2: 20.0: 4.0 1.0: 1.2: 20.0: 5.0 1.0: 1.2: 20.0: 6.0 1.0: 1.2:20.0: 7.0 1.0: 1.2: 20.0: 8.0 1.0: 1.2: 22.0: 6.0 1.0: 1.2: 24.0: 6.0(Y + F = 2.2): 18.0: 6.0 0.66: 1.54: 18.0: 6.0 0.88: 1.32: 18.0: 6.01.1: 1.1: 18.0: 6.0 1.32: 0.88: 18.0: 6.0 1.54: 0.66: 18.0: 6.0 1.0:1.0: 5.0 < 15.0: 6.0 1.0: 1.0: 10.0: 6.0

In a preferred embodiment, the length of any of such copolymer isbetween 35 and 75 amino acids residues. More preferably, the length of arandom copolymer is between 35 and 65 amino acid residues. In apreferred embodiment the length of a random copolymer is about 50 aminoacids. In another preferred embodiment, the length of a random copolymeris about 52 amino acids.

A preferred embodiment of the invention is a random copolymer of thecomposition YFAK in an average molar output ratio of about1.0:1.2:18.0:6.0 respectively, synthesized by solid phase chemistry asdescribed below in more detail.

In a preferred embodiment, the average molar output ratio of YFAK isabout 1.0:1.2:XA:6.0, wherein XA is greater than 18, and the ratio ofalanine increases with the length of copolymer. In a preferredembodiment, the length of such random copolymer is about 52 amino acidresidues, and the ratio of alanine composition in amino acid positions31-52 is greater than in amino acid positions 11-30, and the ratio ofalanine composition in amino acid positions 11-30 is greater than inamino acid positions 1-10. More specifically, a preferred embodiment ofthe invention is a random copolymer of the composition YFAK (L-tyrosine,L-phenylalanine, L-alanine and L-lysine) in an average molar outputratio of about 1.0:1.2:18.0:6.0 respectively, synthesized by solid phasechemistry, wherein the copolymer has a length of 52 amino acids, andwherein residues 1-10 of the copolymer sequence has a molar output ratioof about 1.0:1.2:16:6, residues 11-30 have a molar output ratio of about1.0:1.2:18:6, and residues 31-52 have a molar output ratio of about1.0:1.2:20:6.

Copolymers Comprising Three Amino Acids

In another embodiment, the random copolymer contains three differentamino acids each from a different one of three groups of the abovementioned groups (a) to (d). These copolymers are herein referred to as“terpolymers.” The average molecular weight is between 2,000 to about40,000 daltons, and preferably between about 3,000 to about 35,000daltons. In a more preferred embodiment, the average molecular weight isabout 5,000 to about 25,000 daltons.

In one embodiment, the terpolymers for use in the present inventioncontain tyrosine, alanine, and lysine, hereinafter designated YAK. Theaverage molar fraction of the amino acids in these terpolymers can vary.For example, tyrosine can be present in a mole fraction of about 0.005to about 0.250; alanine can be present in a mole fraction of about 0.3to about 0.6; and lysine can be present in a mole fraction of about 0.1to about 0.5. It is possible to substitute arginine for lysine, glycinefor alanine, and/or tryptophan for tyrosine. The molar ratio of themonomers of the more preferred terpolymer of tyrosine, alanine andlysine, or YAK, is about 0.10 to about 0.54 to about 0.35. Exemplary YAKcopolymers are described in Fridkis-Hareli M., Hum Immunol. 2000; 61(7):640-50.

In another embodiment, the terpolymers for use in the present inventioncontain tyrosine, glutamic acid, and lysine, hereinafter designated YEK.The average molar fraction of the amino acids in these terpolymers canvary: glutamic acid can be present in a mole fraction of about 0.005 toabout 0.300, tyrosine can be present in a mole fraction of about 0.005to about 0.250, and lysine can be present in a mole fraction of about0.3 to about 0.7. It is possible to substitute aspartic acid forglutamic acid, arginine for lysine, and/or tryptophan for tyrosine. Themolar ratios of the monomers of the more preferred terpolymer ofglutamic acid, tyrosine, and lysine, or YEK, is about 0.26 to about 0.16to about 0.58.

In another embodiment the terpolymers for use in the present inventioncontain lysine, glutamic acid, and alanine, hereinafter designated KEA.The average molar fraction of the amino acids in these polypeptides canalso vary. For example, glutamic acid can be present in a mole fractionof about 0.005 to about 0.300, alanine can be present in a mole fractionof about 0.005 to about 0.600, and lysine can be present in a molefraction of about 0.2 to about 0.7. It is possible to substituteaspartic acid for glutamic acid, glycine for alanine, and/or argininefor lysine. The molar ratios of the monomers of the more preferredterpolymer of glutamic acid, alanine and lysine, or KEA, is about 0.15to about 0.48 to about 0.36.

In another embodiment, the terpolymers for use in the present inventioncontain tyrosine, glutamic acid, and alanine, hereinafter designatedYEA. The average molar fraction of the amino acids in these polypeptidescan vary. For example, tyrosine can be present in a mole fraction ofabout 0.005 to about 0.250, glutamic acid can be present in a molefraction of about 0.005 to about 0.300, and alanine can be present in amole fraction of about 0.005 to about 0.800. It is possible tosubstitute tryptophan for tyrosine, aspartic acid for glutamic acid,and/or glycine for alanine. The molar ratios of the monomers of the morepreferred terpolymer of glutamic acid, alanine, and tyrosine, or YEA, isabout 0.21 to about 0.65 to about 0.14.

In a more preferred embodiment, the molar fraction of amino acids of theterpolymers is about what is preferred for Copolymer 1. The molefraction of amino acids in Copolymer 1 is glutamic acid about 0.14,alanine about 0.43, tyrosine about 0.10, and lysine about 0.34. The mostpreferred average molecular weight for Copolymer 1 is between about5,000 and about 9,000 daltons. The activity of Copolymer 1 for theutilities disclosed herein is expected to remain if one or more of thefollowing substitutions is made: aspartic acid (D) for glutamic acid(E), glycine (G) for alanine (A), arginine (R) for lysine (K), andtryptophan (W) for tyrosine (Y).

Copolymers that Bind to MHC Class II Proteins

In one embodiment, the copolymers used in the methods described hereinare capable of binding to an MHC class II protein which, preferably, isassociated with an autoimmune disease. There are at least three types ofClass II MHC molecules: HLA-DR, HLA-DQ, and HLA-DP molecules. There arealso numerous alleles encoding each type of these HLA molecules. TheClass II MHC molecules are expressed predominantly on the surfaces of Blymphocytes and antigen presenting cells such as macrophages. Anyavailable method can be used to ascertain whether the copolymer binds toone or more MHC class II proteins. For example, the polypeptide can belabeled with a reporter molecule (such as a radionuclide or biotin),mixed with a crude or pure preparation of MHC class II protein andbinding is detected if the reporter molecule adheres to the MHC class IIprotein after removal of the unbound polypeptide.

In another embodiment, the copolymers used in the methods describedherein are capable of binding to an MHC class II protein associated withmultiple sclerosis. A polypeptide of this embodiment can have similar orgreater affinity for the antigen binding groove of an MHC class IIprotein associated with multiple sclerosis than does Copolymer 1. Hence,the contemplated polypeptide can inhibit binding of or displace thebinding of myelin autoantigens from the MHC class II protein. One MHCclass II protein associated with multiple sclerosis is HLA-DR4(DRB1*1501).

In another embodiment, the random copolymers used in the methodsdescribed herein are capable of binding to an MHC class II proteinassociated with an arthritic condition, for example, rheumatoidarthritis or osteoarthritis. A random copolymer of this embodiment canhave a greater affinity for the antigen binding groove of an MHC classII protein associated with the autoimmune disease than does a type IIcollagen 261-273 peptide. Hence, the contemplated Copolymer 1 or arandom copolymer described herein such as YFAK can inhibit binding of ordisplace the type II collagen 261-273 peptide from the antigen bindinggroove of an MHC class II protein. The Class II MHC protein consists ofapproximately equal-sized alpha and beta subunits, both of which aretransmembrane proteins. A peptide-binding cleft is formed by parts ofthe amino termini of both α and β subunits. This peptide-binding cleftis the site of presentation of the antigen to T cells.

In other embodiments, the random copolymers used in the invention canbind to the peptide binding groove of the HLA-DR molecules. As bindingmotifs of Cop 1 to MS-associated HLA-DR molecules are known(Fridkis-Hareli et al, 1999, J. Immunol.; 162(8):4697-704), polypeptidesof fixed sequence can readily be prepared and tested for binding to thepeptide binding groove of the HLA-DR molecules as described inFridkis-Hareli. Examples of such peptides are those disclosed in WO00/005249, the entire contents of which being hereby incorporated hereinby reference. Thirty-two of the peptides specifically disclosed in saidapplication are as follows:

AAAYAAAAAAKAAAA (SEQ. ID NO: 1) AEKYAAAAAAKAAAA (SEQ. ID NO: 2)AKEYAAAAAAKAAAA (SEQ. ID NO: 3) AKKYAAAAAAKAAAA (SEQ. ID NO: 4)AEAYAAAAAAKAAAA (SEQ. ID NO: 5) KEAYAAAAAAKAAAA (SEQ. ID NO: 6)AEEYAAAAAAKAAAA (SEQ. ID NO: 7) AAEYAAAAAAKAAAA (SEQ. ID NO: 8)EKAYAAAAAAKAAAA (SEQ. ID NO: 9) AAKYEAAAAAKAAAA (SEQ. ID NO: 10)AAKYAEAAAAKAAAA (SEQ. ID NO: 11) EAAYAAAAAAKAAAA (SEQ. ID NO: 12)EKKYAAAAAAKAAAA (SEQ. ID NO: 13) EAKYAAAAAAKAAAA (SEQ. ID NO: 14)AEKYAAAAAAAAAAA (SEQ. ID NO: 15) AKEYAAAAAAAAAAA (SEQ. ID NO: 16)AKKYEAAAAAAAAAA (SEQ. ID NO: 17) AKKYAEAAAAAAAAA (SEQ. ID NO: 18)AEAYKAAAAAAAAAA (SEQ. ID NO: 19) KEAYAAAAAAAAAAA (SEQ. ID NO: 20)AEEYKAAAAAAAAAA (SEQ. ID NO: 21) AAEYKAAAAAAAAAA (SEQ. ID NO: 22)EKAYAAAAAAAAAAA (SEQ. ID NO: 23) AAKYEAAAAAAAAAA (SEQ. ID NO: 24)AAKYAEAAAAAAAAA (SEQ. ID NO: 25) EKKYAAAAAAAAAAA (SEQ. ID NO: 26)EAKYAAAAAAAAAAA (SEQ. ID NO: 27) AEYAKAAAAAAAAAA (SEQ. ID NO: 28)AEKAYAAAAAAAAAA (SEQ. ID NO: 29) EKYAAAAAAAAAAAA (SEQ. ID NO: 30)AYKAEAAAAAAAAAA (SEQ. ID NO: 31) AKYAEAAAAAAAAAA. (SEQ. ID NO: 32)

Additional random copolymers for use in the present invention, andmethods of synthesizing them, may be found in the literature, such as inShukaliak Quandt, J. et al., 2004, Mol. Immunol. 40(14-15):1075-87;Montaudo, M. S., 2004, J. Am. Soc. Mass Spectrom. 15(3):374-84; Takeda,N. et al., 2004, J. Control Release 95(2): 343-55; Pollino, J. M. etal., 2004, J. Am. Chem. Soc. 126(2):563-7; Fridkis-Hareli, M. et al.,2002, J. Clin Invest. 109(12):1635-43; Williams, D. M. et al., 2000, J.Biol. Chem. 275(49): 38127-30; Tselios, T. et al., 2000, Bioorg. Med.Chem. 8(8): 1903-9; and Cady, C. T. et al., 2000, J. Immunol. 165(4):1790-8.

In one specific embodiment, the random copolymer comprises at leastseven amino acid residues in length and is capable of binding to an MHCclass II protein associated with an autoimmune disease, the syntheticpeptide binding with greater affinity to the antigen binding groove ofthe MHC class II protein than a type II collagen 261-273 peptide,wherein the synthetic peptide comprises a sequence selected from thegroup consisting of alanine-glutamic acid-lysine-tyrosine-alanine(AEKYA) (SEQ ID NO:41), alanine-glutamic acid-lysine-valine-alanine(AEKVA) (SEQ ID NO:42), alanine-glutamicacid-lysine-phenylalanine-alanine (AEKFA) (SEQ ID NO:43),alanine-lysine-tyrosine-alanine-glutamic acid (AKYAE) (SEQ ID NO:44),glutamic acid-alanine-lysine-tyrosine-alanine (EAKYA) (SEQ ID NO:45),alanine-lysine-valine-alanine-glutamic acid (AKVAE) (SEQ ID NO:46), andglutamic acid-alanine-lysine-valine-alanine (EAKVA) (SEQ ID NO:47),alanine-lysine-phenylalanine-alanine-glutamic acid (AKFAE) (SEQ IDNO:48), and glutamic acid-alanine-lysine-phenylalanine-alanine (EAKFA)(SEQ ID NO:49).

In certain preferred embodiments, the copolymers of the invention bindto HLA-DQA1 molecules, and in even more preferably to one or more of HLAmolecules encoded in the alleles DQA1*0501-DQB1*0201, DQA1*0301,DQB1*0401, and DQA1*03-DQB1*0302.

In other embodiments, the copolymers of the methods of the presentinvention bind to certain HLA-DQ molecules that predispose the carrierof such molecules to autoimmune-associated diseases, such as type Idiabetes and celiac disease, with a dissociation constant (K_(d)) atleast 10 times less than the copolymer's K_(d) for binding HLA-DRmolecules and/or other DQ isotypes. Such HLA-DQ molecules are thecombined protein products of specific HLA-DQB1 and DQA1 alleles known asDQB1*0201, DQB1*0302, DQB1*0304, DQB1*0401, DQB1*0501, DQB1*0502; andDQA1*0301, DQA1*0302, DQA1*0303, DQA1*0501. These alleles may be encodedon the same haplotypes (“cis” alleles) such asDQB1*0201-DQA1*0501-DRB1*0301 and DQB1*0302-DQA1*0301-DRB1*0401. Theresulting HLA molecule comprising polypeptide products of “cis” allelesare herein referred to as “cis dimer.” Alternatively, the alleles may beencoded on different haplotypes (“trans” alleles). The HLA moleculecomprising polypeptide products of “trans” alleles are herein referredto as “trans” dimer. An example of “trans” alleles is the combination ofDQB1*0201 on DQB1*0201-DQA1*0501-DRB1*0301 and DQA1*0301 onDQB1*0301-DQA1*0301-DRB1*0404.

In certain embodiments, the DQ-directed copolymers used in the methodsdescribed herein are a mixture of randomized or partially randomizedamino acid sequence containing amino acids from each of the followingfour groups: (1) hydrophobic, aliphatic amino acids (such as leucine,isoleucine, valine, methionine); (2) amino acids with acidic side chains(such as aspartic acid, glutamic acid); (3) amino acids with smallhydrophilic side chains (such as serine, cysteine, threonine); and (4)amino acids with small aliphatic side chains (such as alanine, glycine);additionally, the copolymer contains proline residues. In oneembodiment, the copolymer is derived using the amino acids Glutamine (E)and/or Aspartic acid (D), Leucine (L), Serine (S) and Alanine (A), andis referred to herein as an “ELSA” copolymer.

In certain other embodiments, the DQ-directed copolymers are a mixtureof randomized or partially randomized amino acid sequence containingamino acids from each of the following four groups: (1) hydrophobic,aliphatic amino acids (such as leucine, isoleucine, valine, methionine);(2) bulky hydrophobic amino acids (such as tyrosine, phenylalanine,leucine, methionine); (2) amino acids with acidic side chains (such asaspartic acid, glutamic acid); (3) amino acids with small hydrophilicside chains (such as serine, cysteine, threonine); and (4) amino acidswith small aliphatic side chains (such as alanine, glycine);additionally, the copolymer contains proline residues. An exemplarycopolymer is derived using the amino acid residues Glutamine (E) and/orAspartic acid (D), Leucine (L), Tyrosine (Y) and Val (V), and isreferred to herein as an “DLYV” copolymer.

In one embodiment, a method of treatment of an autoimmune diseasecomprises administration of a copolymer that binds to an HLA-DQ moleculeassociated with the autoimmune disease. Preferably, the method oftreatment is carried out using a copolymer that comprises a polypeptidecomprising a plurality of amino acid residues selected from: (1) ahydrophobic, aliphatic residue (leucine, isoleucine, valine,methionine); (2) an acidic residue (aspartic acid, glutamic acid); (3) asmall hydrophilic residue (serine, cysteine, threonine); (4) a smallaliphatic residue (alanine, glycine); and (5) proline.

In preferred embodiments, the copolymers compositions of the presentinvention bind to one or more DQ isotypes with an average K_(d) of 1 μMor less, and more preferably an average K_(d) less than 100 nM, 10 nM oreven 1 nM. Another way to identify preferred copolymers is based on themeasure of a copolymer to displace another in competitive bindingassays, such as described in Sidney et al., 2002, J. Immunol. 169:5098,which is expressed as an IC₅₀ value. Preferred copolymers of the presentinvention have IC₅₀'s less than 1 μM, more preferably less than 500 nM,and even more less than 100 nM.

In certain preferred embodiments, the copolymer is formed by randomsynthesis (polymerization) of the various amino acid residues. A certainratio of amino acids to be incorporated into the random copolymer may beused. Preferred random copolymers of the present invention compriseamino acid residues K, E, A, S, V, and P. More preferably, the ratio ofK:E:A:S:V is 0.3:0.7:9:0.5:0.5:0.3. Preferably, the random copolymersare about 10 to 100 amino acid residues long, more preferably 20 to 80amino acid residues long, even more preferably 40 to 60 amino acidresidues long, and most preferably about 50 amino acid residues long.When synthesized, a typical preparation of random copolymers is amixture of peptides of various lengths, the majority of which are of thedesired length but containing shorter or longer peptides inevitablycreated by the currently available synthetic processes.

Further, in certain embodiments, the copolymer can be a semi-random (orsemi-regular) polymer having “anchor,” or fixed, residues which occurwith regular spacing in the resulting polymer, providing for optimalclass II binding. The anchor residues within the peptide may be E, D, orV. For example, the copolymer can be synthesized to have one of thegeneral sequences:

1. [XXEXXXXXXXEXX]_(n) (SEQ. ID NO: 33) 2. [XXEXXXXXXXDXX]_(n) (SEQ. IDNO. 34) 3. [XXDXXXXXXXDXX]_(n) (SEQ. ID NO. 35) 4. [XXDXXXXXXXEXX]_(n)(SEQ. ID NO. 36) 5. [XXEXXVXXXXDXX]_(n) (SEQ. ID NO. 37) 6.[XXDXXVXXXXDXX]_(n) (SEQ. ID NO. 38) 7. [XXDXXVXXXXEXX]_(n) (SEQ. ID NO.39) 8. [XXEXXVXXXXEXX]_(n) (SEQ. ID NO. 40) wherein X is A, S, V, K, orP, the ratio of which are 5:1:1:1:0.5, and 1 ≦ n ≦ 8.

The peptides may have a length of 9 to 25 amino acid residues.Preferably, the peptide is 13 amino acid-residues long. A peptide of adefined sequence length of 9 to 25 amino acids may contain from 2 to 20fixed residues. An individual fixed residue of a peptide described inthis invention may bind to the peptide binding grove of a class II MCHmolecule at any of the positions P1, P4, P7, or P9. Preferably, suchpeptide contains 2 or 3 fixed residues. In one embodiment, a peptide ofa defined sequence length of 13 amino acids will contain 2 fixedresidues, either E or D or any combination thereof. Preferably a peptideof a defined sequence length of 13 amino acids will contain 3 fixedresidues. The peptides may be multimers of a defined sequence, whereinthe number of the repeating units preferably ranges from 2 to 8. Morepreferably, the number of the repeating units is 3 to 6. Mostpreferably, the number of repeating units is 4. In a preferredembodiment, a multimer of the instant invention comprises a peptide of adefined sequence length of 13 amino acids containing 2 fixed residues,either E or D or any combination thereof.

In certain preferred embodiments, the subject copolymers are formulatedfor use as a medicament so as to have a polydispersity less than 25,000,and more preferably less than 10000, 5000, 1000, 500, 100, 50, or evenless than 10.

Synthesis of Random Copolymers

The terpolymers and random copolymers used in the present invention canbe made by any procedure available to one of skill in the art. Forexample, the terpolymers can be made under condensation conditions usingthe desired molar ratio of amino acids in solution, or by solid phasesynthetic procedures. Condensation conditions include the propertemperature, pH, and solvent conditions for condensing the carboxylgroup of one amino acid with the amino group of another amino acid toform a peptide bond. Condensing agents, for exampledicyclohexyl-carbodiimide, can be used to facilitate the formation ofthe peptide bond. Blocking groups can be used to protect functionalgroups, such as the side chain moieties and some of the amino orcarboxyl groups against undesired side reactions.

For example, the process disclosed in U.S. Pat. No. 3,849,550, can beused wherein the N-carboxyanhydrides of tyrosine, alanine, γ-benzylglutamate and N-ε-trifluoroacetyl-lysine are polymerized at ambienttemperatures in anhydrous dioxane with diethylamine as an initiator. Theγ-carboxyl group of the glutamic acid can be deblocked by hydrogenbromide in glacial acetic acid. The trifluoroacetyl groups are removedfrom lysine by 1 molar piperidine. One of skill in the art readilyunderstands that the process can be adjusted to make peptides andpolypeptides containing the desired amino acids, that is, three of thefour amino acids in Copolymer 1, by selectively eliminating thereactions that relate to any one of glutamic acid, alanine, tyrosine, orlysine. For purposes of this application, the terms “ambienttemperature” and “room temperature” mean a temperature ranging fromabout 20 to about 26° C.

A preferred synthesis method of the random copolymers of the presentinvention is by solid phase synthesis. The synthesis is done in multiplesteps by the Solid Phase Peptide Synthesis (SPPS) approach using Fmocprotected amino acids. SPPS is based on sequential addition of protectedamino acid derivatives, with side chain protection where appropriate, toa polymeric support (bead). The base-labile Fmoc group is used forN-protection. After removing the protecting group (via piperidinehydrolysis) the next amino acid mixture is added using a couplingreagent (TBTU). After the final amino acid is coupled, the N-terminus isacetylated.

The resulting peptide (attached to the polymeric support through itsC-terminus) is cleaved with TFA to yield the crude peptide. During thiscleavage step, all of the side chains protecting groups are alsocleaved. After precipitation with diisopropyl ether, the solid isfiltered and dried. The resulting peptide is analyzed and stored at 2-8°C.

Example of Solid Phase Synthesis

The random copolymer YFAK consisting of L-alanine, L-lysine,L-phenylalanine and L-tyrosine is prepared in its protected form on Wangresin. Resins used were Fmoc-L-Tyr(t-Bu)-Wang (0.62 mmol/g),Fmoc-L-Phe-Wang (0.72 mmol/g), Fmoc-L-Ala-Wang (0.70 mmol/g), andFmoc-L-Lys(Boc)-Wang (0.72 mmol/g). The four F-moc protected aminoacids, Fmoc-L-Tyr(t-Bu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Ala-OH, andFmoc-L-Lys-OH, are used in a molar input ratio of 1:1:10:6 respectivelyduring each coupling step. Other reagents used in the synthesis are2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium, tetrafluoroborate(TBTU), N,N-diisopropylethylamine (DIPEA), piperidine, andtrifluoroacetic acid (TFA). The solvents used are N-methylpyrrolidone(NMP), isopropanol (IsOH, IPA, i-PrOH), methylene chloride, andisopropyl ether. The stoichiometry of each coupling is as follows:

-   -   residues 1 through 10 using 2 equivalents of Fmoc protected        amino acids;    -   residues 11 through 30 using 2 equivalents with double coupling        of Fmoc protected amino acids;    -   residues 31 through 52 using 2.5 equivalents of Fmoc protected        amino acids with double coupling.

An example of amino acid input ratios in a representative example ofYFAK synthesis with progressively higher alanine contents is as follows:

Positions Y F A K  0-10 3.7 5.5 64.4 26.4 11-20 4.3 5.1 71.4 19.2 21-304.0 4.7 71.5 19.8 31-40 3.6 4.7 74.3 17.4 41-52 3.0 4.1 76.0 16.8

In a similar manner, Copolymer 1, a random copolymer of a preferredembodiment of the invention, is prepared in its protected form on Wangresin. Resins used were Fmoc-L-Tyr(t-Bu)-Wang (0.62 mmol/g),Fmoc-L-Glu-Wang, Fmoc-L-Ala-Wang (0.70 mmol/g), and Fmoc-L-Lys(Boc)-Wang(0.72 mmol/g). The four F-moc protected amino acids,Fmoc-L-Tyr(t-Bu)-OH, Fmoc-L-Glu-OH, Fmoc-L-Ala-OH, and Fmoc-L-Lys-OH,are used in a molar input ratio of 1:2:6:5 respectively during eachcoupling step. Other reagents used and coupling stoichiometry is as withthe synthesis of YFAK.

An example of amino acid input ratios in a representative example ofYEAK synthesis with progressively higher alanine contents is as follows:

Positions Y E A K  0-10 3.7 9.1 21.4 22.0 11-20 4.3 8.5 23.8 16.0 21-304.0 8.0 23.9 16.5 31-40 3.6 7.8 24.8 14.5 41-52 3.0 6.8 25.3 14.0Unnatural Polypeptides and Chemical Modification of Copolymers

In one embodiment, the copolymers of the present invention are composedof naturally-occurring amino acids. In other embodiments, the copolymersare comprised of naturally occurring and synthetic derivatives, forexample, selenocysteine. Amino acids further include amino acid analogs.An amino acid “analog” is a chemically related form of the amino acidhaving a different configuration, for example, an isomer, or aD-configuration rather than an L-configuration, or an organic moleculewith the approximate size and shape of the amino acid, or an amino acidwith modification to the atoms that are involved in the peptide bond, soas to be protease resistant when polymerized in a polypeptide.

The copolymers for use in the present invention can be composed of L- orD-amino acids or mixtures thereof. As is known by those of skill in theart, L-amino acids occur in most natural proteins. However, D-aminoacids are commercially available and can be substituted for some or allof the amino acids used to make the terpolymers and other copolymers ofthe present invention. The present invention contemplates copolymerscontaining both D- and L-amino acids, as well as copolymers consistingessentially of either L- or D-amino acids.

In certain embodiments, the random copolymers of the present inventioninclude such linear copolymers that are further modified by substitutingor appending different chemical moieties. In one embodiment, suchmodification is at a residue location and in an amount sufficient toinhibit proteolytic degradation of the copolymer in a subject. Forexample, the amino acid modification may be the presence in the sequenceof at least one proline residue; the residue is present in at least oneof carboxy- and amino termini; further, the proline can be presentwithin four residues of at least one of the carboxy- and amino-termini.Further, the amino acid modification may be the presence of a D-aminoacid.

In certain embodiments, the subject random copolymer is apeptidomimetic. Peptidomimetics are compounds based on, or derived from,peptides and proteins. The copolymer peptidomimetics of the presentinvention typically can be obtained by structural modification of one ormore native amino acid residues, e.g., using unnatural amino acids,conformational restraints, isosteric replacement, and the like. Thesubject peptidomimetics constitute the continuum of structural spacebetween peptides and non-peptide synthetic structures.

Such peptidomimetics can have such attributes as being non-hydrolyzable(e.g., increased stability against proteases or other physiologicalconditions which degrade the corresponding peptide copolymers),increased specificity and/or potency. For illustrative purposes, peptideanalogs of the present invention can be generated using, for example,benzodiazepines (e.g., see Freidinger et al. in “Peptides: Chemistry andBiology,” G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), substituted gamma lactam rings (Garvey et al. in “Peptides:Chemistry and Biology,” G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988, p 123), C-7 mimics (Huffman et al. in “Peptides:Chemistry and Biology,” G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988, p. 105), keto-methylene pseudopeptides (Ewenson etal., 1986, J. Med. Chem. 29:295; and Ewenson et al. in “Peptides:Structure and Function (Proceedings of the 9th American PeptideSymposium),” Pierce Chemical Co. Rockland, Ill., 1985), β-turn dipeptidecores (Nagai et al., 1985, Tetrahedron Lett. 26:647; and Sato et al.,1986, J. Chem. Soc. Perkin Trans. 1:1231), β-aminoalcohols (Gordon etal., 1985, Biochem. Biophys. Res. Commun. 126:419; and Dann et al.,1986, Biochem. Biophys. Res. Commun. 134:71), diaminoketones (Natarajanet al., 1984, Biochem. Biophys. Res. Commun. 124:141), andmethyleneamino-modified (Roark et al. in “Peptides: Chemistry andBiology,” G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988, p 134). Also, see generally, Session III: Analytic and syntheticmethods, in “Peptides: Chemistry and Biology,” G. R. Marshall ed., ESCOMPublisher: Leiden, Netherlands, 1988)

The molecular weight of a random copolymer can be adjusted duringpolypeptide synthesis or after the copolymer have been synthesized. Toadjust the molecular weight during polypeptide synthesis, the syntheticconditions or the amounts of amino acids are adjusted so that synthesisstops when the polypeptide reaches the approximate length which isdesired. After synthesis, polypeptides with the desired molecular weightcan be obtained by any available size selection procedure, such aschromatography of the polypeptides on a molecular weight sizing columnor gel, and collection of the molecular weight ranges desired. Thepresent polypeptides can also be partially hydrolyzed to remove highmolecular weight species, for example, by acid or enzymatic hydrolysis,and then purified to remove the acid or enzymes.

In one embodiment, the random copolymers with a desired molecular weightmay be prepared by a process which includes reacting a protectedpolypeptide with hydrobromic acid to form a trifluoroacetyl-polypeptidehaving the desired molecular weight profile. The reaction is performedfor a time and at a temperature which is predetermined by one or moretest reactions. During the test reaction, the time and temperature arevaried and the molecular weight range of a given batch of testpolypeptides is determined. The test conditions which provide theoptimal molecular weight range for that batch of polypeptides are usedfor the batch. Thus, a trifluoroacetyl-polypeptide having the desiredmolecular weight profile can be produced by a process which includesreacting the protected polypeptide with hydrobromic acid for a time andat a temperature predetermined by test reaction. Thetrifluoroacetyl-polypeptide with the desired molecular weight profile isthen further treated with an aqueous piperidine solution to form a lowtoxicity polypeptide having the desired molecular weight.

In one preferred embodiment, a test sample of protected polypeptide froma given batch is reacted with hydrobromic acid for about 10-50 hours ata temperature of about 20-28° C. The best conditions for that batch aredetermined by running several test reactions. For example, in oneembodiment, the protected polypeptide is reacted with hydrobromic acidfor about 17 hours at a temperature of about 26° C.

In some embodiments, random copolymers which may be used in theinvention include those described in International PCT Publication Nos.WO 00/05250, WO 00/05249; WO 02/59143, WO 0027417, WO 96/32119, in U.S.Patent Publication Nos. 2004/003888, 2002/005546, 2003/0004099,2003/0064915 and 2002/0037848, in U.S. Pat. Nos. 6,514,938, 5,800,808and 5,858,964, and those described in PCT application PCT/US05/06822.These references further describe methods of synthesizing randomcopolymers, compositions comprising random copolymers, therapeuticformulations of random copolymers, methods of administering randomcopolymers to a subject, diseases that may be treated with randomcopolymers, and additional therapeutically effective agents which may beco-administered to a subject in with the random copolymers. Theteachings of all these patents, applications and publications are hereinincorporated by reference in their entirety.

It is clear that this is given by way of example only, and that thecomposition can be varied both with respect to the constituents andrelative proportions of the constituents if the above general criteriaare adhered to.

IV. Diseases

The invention provides methods for treating or preventing diseases in asubject. A subject who is at risk of developing a disease, who issuspected of being afflicted with a disease, or who is afflicted withthe disease may be treated using the methods provided by the invention.

In one embodiment, the disease that may be treated with the methods ofthe present invention comprises a disease that is mediated by T-cells,and in particular T_(H)1 cells, or is a disease which is exacerbated byan excess of inflammatory cytokines. The methods of the presentinvention may be used to treat diseases comprising ischemic injuries,including those caused by systemic ischemia or local ischemia,particularly to the heart, lungs or kidneys. In some embodiments, theinflammation is associated with a septic shock, anaphylactic shock,toxic shock syndrome, cachexia, necrosis, gangrene, a prostheticimplant, or hypersensitivity, including Type I hypersensitivity, Type IIhypersensitivity, Type III hypersensitivity, Type IV hypersensitivity,immediate hypersensitivity, antibody mediated hypersensitivity, immunecomplex mediated hypersensitivity, T lymphocyte mediatedhypersensitivity and delayed type hypersensitivity. In otherembodiments, the disease comprises myocardial infarction, cardiacarrest, ischemia-reperfusion injury, congestive heart failure,cardiotoxicity, cardiac damage due to parasitic infection, fulminantcardiac amyloidosis, heart surgery, heart transplantation, traumaticcardiac injury, surgical repair of a thoracic aortic aneurysm, asuprarenal aortic aneurysm, hemorrhagic shock due to blood loss,cardiogenic shock due to myocardial infarction or cardiac failure,anaphylaxis, unstable coronary syndrome, tachycardia, bradycardia or acombination thereof.

In one embodiment, the disease that may be treated with the methods ofthe present invention comprises autoimmune diseases. Autoimmune diseasescontemplated by the present invention include either cell-mediateddisease (e.g., T-cell) or antibody-mediated (e.g., B cell) disorders.Such disorders can be inter alia arthritic conditions, demyelinatingdiseases and inflammatory diseases. The methods of the invention are ofparticular interest for the treatment of demyelinating inflammatorydiseases, which include multiple sclerosis, EAE, optic neuritis, acutetransverse myelitis, and acute disseminated encephalitis. In onespecific embodiment, any autoimmune disease can be treated by thepresent polypeptides so long as the contemplated polypeptide binds to anMHC class II protein that has been associated with the autoimmunedisease. Progression of disease can be measured by monitoring clinicalor diagnostic symptoms using known methods.

In one embodiment, the disease treated by the methods provided herein isan “arthritic condition”. As used herein, an arthritic condition is acondition wherein at least one symptom of rheumatoid arthritis isobserved in at least one joint of a mammal, for example in a shoulder,knee, hip, backbone or a digit of the mammal. RA is a common humanautoimmune disease with a prevalence of about 1% among Caucasians(Harris, B. J. et al., 1997, In Textbook of Rheumatology 898-932),currently affecting 2.5 million Americans. RA is characterized bychronic inflammation of the synovial joints and infiltration byactivated T cells, macrophages and plasma cells, leading to aprogressive destruction of the articular cartilage. It is the mostsevere form of joint disease. Inherited susceptibility to RA is stronglyassociated with the affected subject having at the MHC class II DRB1locus the allele DRB1*0401, DRB1*0404, or DRB1*0405 or the DRB1*0101allele. The nature of the autoantigen(s) in RA is poorly understood,although collagen type II (CII) is a prominent candidate. Animmunodominant T cell epitope in collagen type II corresponding toresidues 261-273 has been identified (Fugger, L. et al., 1996, Eur. J.Immunol. 26: 928-933).

Other examples of arthritic conditions include “polyarthritis”, which isan arthritic condition that affects more than a single joint; “juvenilearthritis”, an arthritic condition of humans under the age of 21; andFelty's syndrome, which can include the symptoms of neutropenia,splenomegaly, weight loss, anemia, lymphadenopathy, and pigment spots onthe skin.

In another embodiment, the disease treated by the methods providedherein is multiple sclerosis (MS). The course of disease for multiplesclerosis is highly varied, unpredictable, and, in most patients,remittent. The pathologic hallmark of MS is multicentric, multiphasicCNS inflammation and demyelination. Months or years of remission mayseparate episodes, particularly early in the disease. About 70% ofpatients of relapsing-remitting (RR) type, which is characterized byacute exacerbations with full or partial remissions. The remainingpatients present with chronic progressive MS, which is subdividedfurther into (a) primary-progressive (PP), (b) relapsing-progressive(RP), which is a pattern combining features of RR and RP and isintermediate in clinical severity, and (c) secondary-progressive (SP),which many patients with RR progress to over time. In a specificpreferred embodiment, the disease treated by the present method isrelapsing-remitting multiple sclerosis.

Clinical symptoms of MS include sensory loss (paresthesias), motor(muscle cramping secondary to spasticity) and autonomic (bladder, bowel,sexual dysfunction) spinal cord symptoms; cerebellar symptoms (e.g.,Charcot triad of dysarthna, ataxia, tremor); fatigue and dizziness;impairment in information processing on neuropsychological testing; eyesymptoms, including diplopia on lateral gaze; trigeminal neuralgia; andoptic neuritis.

The autoantigen in MS most likely is one of several myelin proteins(e.g, proteolipid protein (PLP); myelin oligodendrocyte glycoprotein(MOG); myelin basic protein (MBP); myelin-associated glycoprotein (MAG),myelin-associated oligodendrocytic basic protein (MBOP);citrulline-modified MBP (the C8 isoform of MBP in which 6 arginines havebeen de-iminated to citrulline), cyclic nucleotide phosphodiesterase(CNPase), alpha-B crystalline, etc.) The integral membrane protein PLPis a dominant autoantigen of myelin. Microglial cells and macrophagesperform jointly as antigen-presenting cells, resulting in activation ofcytokines, complement, and other modulators of the inflammatory process,targeting specific oligodendroglia cells and their membrane myelin. Aquantitative increase in myelin-autoreactive TH1 cells with the capacityto secrete IFN-γ is associated with the pathogenesis of MS and EAE,suggesting that autoimmune inducer/helper T lymphocytes in theperipheral blood of MS patients may initiate and/or regulate thedemyelination process in patients with MS. On the other hand, there isan extended literature on the protective role of TH2 cells producinganti-inflammatory cytokines such as IL-4 and IL-10. The shift of balancefrom TH1 to TH2 type of cells is expected to be beneficial to theprevention and treatment of MS and EAE.

In another embodiment, the disease treated by the methods providedherein is Insulin Dependent Diabetes Mellitus. Human type I orinsulin-dependent diabetes mellitus (IDDM) is characterized byautoimmune destruction of the cells in the pancreatic islets ofLangerhans. The depletion of β-cells results in an inability to regulatelevels of glucose in the blood. Overt diabetes occurs when the level ofglucose in the blood rises above a specific level, usually about 250mg/dl. In humans a long pre-symptomatic period precedes the onset ofdiabetes. During this period there is a gradual loss of pancreatic betacell function. The development of disease is implicated by the presenceof autoantibodies against insulin, glutamic acid decarboxylase, and thetyrosine phosphatase IA2 (IA2), each an example of a self-protein,-polypeptide or -peptide according to this invention. Human IDDM iscurrently treated by monitoring blood glucose levels to guide injection,or pump-based delivery, of recombinant insulin. Diet and exerciseregimens contribute to achieving adequate blood glucose control.

Markers that may be evaluated during the pre-symptomatic stage are thepresence of insulitis in the pancreas, the level and frequency of isletcell antibodies, islet cell surface antibodies, aberrant expression ofClass II MHC molecules on pancreatic beta cells, glucose concentrationin the blood, and the plasma concentration of insulin. An increase inthe number of T lymphocytes in the pancreas, islet cell antibodies andblood glucose is indicative of the disease, as is a decrease in insulinconcentration.

The presence of combinations of autoantibodies with variousspecificities in serum is highly sensitive and specific for human type Idiabetes mellitus. For example, the presence of autoantibodies againstGAD and/or IA-2 is approximately 98% sensitive and 99% specific foridentifying type I diabetes mellitus from control serum. In non-diabeticfirst degree relatives of type I diabetes patients, the presence ofautoantibodies specific for two of the three autoantigens including GAD,insulin and IA-2 conveys a positive predictive value of >90% fordevelopment of type I DM within 5 years.

In another embodiment, the disease treated by the methods providedherein is autoimmune uveitis. Autoimmune uveitis is an autoimmunedisease of the eye that is estimated to affect 400,000 people, with anincidence of 43,000 new cases per year in the U.S. Autoimmune uveitis iscurrently treated with steroids, immunosuppressive agents such asmethotrexate and cyclosporin, intravenous immunoglobulin, andTNFα-antagonists.

In another embodiment, the disease treated by the methods providedherein is experimental autoimmune uveitis (EAU). EAU is a Tcell-mediated autoimmune disease that targets neural retina, uvea, andrelated tissues in the eye. EAU shares many clinical and immunologicalfeatures with human autoimmune uveitis, and is induced by peripheraladministration of uveitogenic peptide emulsified in Complete Freund'sAdjuvant (CFA).

In another embodiment, the disease treated by the methods providedherein is primary biliary cirrhosis (PBC). PBC is an organ-specificautoimmune disease that predominantly affects women between 40-60 yearsof age. The prevalence reported among this group approaches 1 per 1,000.PBC is characterized by progressive destruction of intrahepatic biliaryepithelial cells (IBEC) lining the small intrahepatic bile ducts. Thisleads to obstruction and interference with bile secretion, causingeventual cirrhosis. Association with other autoimmune diseasescharacterized by epithelium lining/secretory system damage has beenreported, including Sjogren's Syndrome, CREST Syndrome, autoimmunethyroid disease and rheumatoid arthritis.

In another embodiment, the disease treated by the methods providedherein is celiac disease, also known as celiac sprue or gluten-sensitiveenteropathy. Celiac disease is a disease that results from defectivegastrointestinal absorption due to hypersensitivity to cereal grainstorage proteins, including glutens or its product gliadin and glutenin,present in wheat, barley, and oats. The disease is caused by CD4 T cellsthat recognize gliadin as dietary antigen and these cells induce aTh1-mediated chronic inflammatory response. Symptoms include diarrhea,weight loss, and steatorrhea, villous atrophy and malabsorption areseen. It may also be associated with dermatitis herpetiforms, avesicular skin eruption. Celiac disease is associated with allelesDQB1*0302 and DQB1*0201 combined with DQA1*0301 and DQA1*0501. 95% ofpatients carry either DQB1*0201 or DQB1*0302. The strong HLA associationis believed to be due to the capacity of DQ molecules encoded byDQB1*0201, DQA1*0501, DQB1*0302 and DQA1*0301 to efficiently presentdeaminated variants of glutamine-rich peptides derived from gliadin andglutenin.

In another embodiment, the method for treating an autoimmune disease ina subject further involves inhibiting the proliferation or function of Tcells which are responsive to an autoantigen. The pathological processof autoimmune diseases and immune rejection is mediated by T cells. Uponbinding to and recognition of an antigen, T cells proliferate, secretecytokines and recruit additional inflammatory and cytotoxic cells to thesite.

In yet another embodiment, the methods described herein for treating anautoimmune disease in a subject involve binding the random copolymer toa major histocompatibility complex class II protein which is associatedwith an autoimmune disease. The Class II MHC proteins are expressedpredominantly on the surfaces of B lymphocytes and antigen presentingcells such as macrophages. These Class II MHC proteins have apeptide-binding cleft which is the site at which antigenic peptides arepresented to T cells. When the present random copolymers bind to a majorhistocompatibility complex class II protein, those random copolymers canblock or otherwise interfere with antigen presentation and/or T cellactivation.

In one embodiment, the disease treated by the methods of the presentinvention is host-versus-graft disease (HVGD) or graft-versus-hostdisease (GVHD). Transplantation systems such as organ transplantationsand bone marrow reconstitution have become important and effectivetherapies for many life threatening diseases. However, immune rejectionis still the major barrier for successful transplantation. This ismanifested in functional deterioration and graft rejection in the caseof organ transplantation (host-versus-graft disease, or HVGD. Anothermanifestation of pathological immune reactivity is GVHD that occurs inapproximately 30% of bone marrow recipients. Up to half of thosepatients who develop GVHD may succumb to this process. This highmorbidity and mortality has led to continuous interest in thepossibility of controlling or preventing GVHD. Clinicopathologically,two forms of GVHD have been recognized. Acute GVHD develops within thefirst 3 months after bone marrow transplantation and features disordersof skin, liver and gastrointestinal tract. Chronic GVHD is a multi-organautoimmune-like disease emerging from 3 months up to 3 yearspost-transplantation and shares features common to naturally occurringautoimmune disorders, like systemic lupus erythematosus (SLE) andscleroderma. The methods described herein may be used to treat bothacute and chronic GVHD.

In a specific embodiment of the methods described herein, Copolymer 1 orYFAK random copolymer may be used for prevention and treatment of GVHDin all cases of organ transplantation that develop GVHD, butparticularly in fetal thymus, and more particularly, in allogeneic bonemarrow, transplantation. To a patient under suitable conditioningregimen, the GLAT copolymer may be administered in a treatment regimenfrom day −2 prior to the transplantation day, and then for another60-100, at least 60 days, after the transplantation day. A regimen ofsuch duration may comprise administrations of the random copolymer atintervals greater than 24, 30, 36, 42, or 48 hours. Otherimmunosuppressive drugs, such as cyclosporine, methotrexate andprednisone, may be administered with the Copolymer 1 copolymer.

The method of the invention may also be applied to the prevention andtreatment of GVHD in the course of bone marrow transplantation inpatients suffering from diseases curable by bone marrow transplantation,including leukemias, such as acute lymphoblastic leukemia (ALL), acutenonlymphoblastic leukemia (ANLL), acute myelocytic leukemia (AML) andchronic myelocytic leukemia (CML), severe combined immunodeficiencysyndromes (SCID), osteopetrosis, aplastic anemia, Gaucher's disease,thalassemia and other congenital or genetically-determined hematopoieticor metabolic abnormalities.

In another embodiment, the methods of the present invention may beapplied to promote nerve regeneration or to prevent or inhibit secondarydegeneration which may otherwise follow primary nervous system injury,e.g., closed head injuries and blunt trauma, such as those caused byparticipation in dangerous sports, penetrating trauma, such as gunshotwounds, hemorrhagic stroke, ischemic stroke, glaucoma, cerebralischemia, or damages caused by surgery such as tumor excision. Inaddition, such compositions may be used to ameliorate the effects ofdisease that result in a degenerative process, e.g., degenerationoccurring in either gray or white matter (or both) as a result ofvarious diseases or disorders, including, without limitation: diabeticneuropathy, senile dementias, Alzheimer's disease, Parkinson's Disease,facial nerve (Bell's) palsy, glaucoma, Huntington's chorea, amyotrophiclateral sclerosis (ALS), status epilepticus, non-arteritic opticneuropathy, intervertebral disc herniation, vitamin deficiency, priondiseases such as Creutzfeldt-Jakob disease, carpal tunnel syndrome,peripheral neuropathies associated with various diseases, including butnot limited to, uremia, porphyria, hypoglycemia, Sjorgren Larssonsyndrome, acute sensory neuropathy, chronic ataxic neuropathy, biliarycirrhosis, primary amyloidosis, obstructive lung diseases, acromegaly,malabsorption syndromes, polycythemia vera, IgA and IgG gammapathies,complications of various drugs (e.g., metronidazole) and toxins (e.g.,alcohol or organophosphates), Charcot-Marie-Tooth disease, ataxiatelangectasia, Friedreich's ataxia, amyloid polyneuropathies,adrenomyeloneuropathy, Giant axonal neuropathy, Refsum's disease,Fabry's disease, lipoproteinemia, etc. In addition, other clinicalconditions that may be treated through the administration of a randomcopolymer in accordance with the present invention include epilepsy,amnesia, anxiety, hyperalgesia, psychosis, seizures, abnormally elevatedintraocular pressure, oxidative stress, and opiate tolerance anddependence.

In a specific embodiments, the disease treated by the methods describedherein comprises multiple sclerosis, type-I diabetes, Hashimoto'sthyroiditis, Crohn's disease, rheumatoid arthritis, systemic lupuserythematosus (SLE), gastritis, autoimmune hepatitis, hemolytic anemia,autoimmune hemophilia, autoimmune lymphoproliferative syndrome (ALPS),autoimmune uveoretinitis, glomerulonephritis, Guillain-Barré syndrome,psoriasis, myasthenia gravis, autoimmune encephalomyelitis,Goodpasture's syndrome, Grave's disease, paraneoplastic pemphigus,autoimmune thrombocytopenic purpura, scleroderma with anti-collagenantibodies, mixed connective tissue disease, pernicious anemia,polymyositis, idiopathic Addison's disease, autoimmune-associatedinfertility, bullous pemphigoid, Sjogren's syndrome, idiopathic myxedemaor colitis. In some embodiments, the subject is afflicted with more thanone disease.

V. Therapeutic Compositions

The random copolymers of the present invention may be administered tothe subject as a composition which comprises a pharmaceuticallyeffective amount of copolymer and an acceptable carrier and/orexcipients. A pharmaceutically acceptable carrier includes any solvents,dispersion media, or coatings that are physiologically compatible.Preferably, the carrier is suitable for intravenous, intramuscular,oral, intraperitoneal, intradermal, transdermal, topical, orsubcutaneous administration. One exemplary pharmaceutically acceptablecarrier is physiological saline. Other pharmaceutically acceptablecarriers and their formulations are well-known and generally describedin, for example, Remington's Pharmaceutical Science (18^(th) Ed., ed.Gennaro, Mack Publishing Co., Easton, Pa., 1990). Variouspharmaceutically acceptable excipients are well-known in the art and canbe found in, for example, Handbook of Pharmaceutical Excipients (4^(th)ed., Ed. Rowe et al. Pharmaceutical Press, Washington, D.C.). Thecomposition can be formulated as a solution, microemulsion, liposome,capsule, tablet, or other suitable forms. The active component whichcomprises the copolymer may be coated in a material to protect it frominactivation by the environment prior to reaching the target site ofaction. The pharmaceutical compositions of the present invention arepreferably sterile and non-pyrogenic at the time of delivery, and arepreferably stable under the conditions of manufacture and storage.

In other embodiments of the present invention, the pharmaceuticalcompositions are regulated-release formulations. Copolymers of thepresent invention may be admixed with biologically compatible polymersor matrices which control the release rate of the copolymers into theimmediate environment. Controlled or sustained release compositionsinclude formulation in lipophilic depots (e.g., fatty acids, waxes,oils).

In some embodiments of the present invention, pharmaceuticalcompositions comprise random copolymers formulated with oil andemulsifier to form water-in-oil microparticles and/or emulsions. The oilmay be any non-toxic hydrophobic material liquid at ambient temperatureto about body temperature, such as edible vegetable oils includingsafflower oil, soybean oil, corn oil, and canola oil; or mineral oil.Chemically defined oil substance such as lauryl glycol may also be used.The emulsifier useful for this embodiment includes Span 20 (sorbitanmonolaurate) and phosphatidylcholine. In some embodiments, a randomcopolymer composition is prepared as an aqueous solution and is preparedinto an water-in-oil emulsion dispersed in 95 to 65% oil such as mineraloil, and 5 to 35% emulsifier such as Span 20. In another embodiment ofthe invention, the emulsion is formed with alum rather than with oil andemulsifier. These emulsions and microparticles reduce the speed ofuptake of random copolymer, and achieve controlled antigen delivery.

In some embodiments, the pharmaceutical compositions also includeadditional therapeutically active agents. Such additional ingredient canbe at least an additional random copolymer, such as a Copolymer 1 (YEAK,Copaxone™) that binds to a different HLA molecule, an antibody whichbinds to an unwanted inflammatory molecule or cytokine such asinterleukin-6, interleukin-8, granulocyte macrophage colony stimulatingfactor, and tumor necrosis factor-α; an enzyme inhibitor such as aprotease inhibitor aprotinin or a cyclooxygenase inhibitor; anantibiotic such as amoxicillin, rifampicin, erythromycin; an antiviralagent such as acyclovir; a steroidal anti-inflammatory such as aglucocorticoid; a non-steroidal anti-inflammatory such as aspirin,ibuprofen, or acetaminophen; or a non-inflammatory cytokine such asinterleukin-4 or interleukin-10. Other cytokines and growth factors suchas interferon-β, tumor necrosis factors, antiangiogenic factors,erythropoietins, thrombopoietins, interleukins, maturation factors,chemotactic protein, and their variants and derivatives that retainsimilar physiological activities may also be used as an additionalingredient.

In some embodiments, the additional active therapeutically active agentis selected from the group consisting of anti-psoriasis creams, eyedrops, nose drops, Sulfasalazine, glucocorticoids, propylthiouracil,methimazole, I¹³¹, insulin, IFN-β1a, IFN-β1b, glucocorticoids, ACTH,avonex, azathiopurine, cyclophosphamide, UV-B, PUVA, methotrexate,calcipitriol, cyclophosphamide, OKT3, FK-506, cyclosporin A,azathioprine, and mycophenolate mofetil.

Copolymers of the invention may also be used in combination withanti-obesity drugs. Anti-obesity drugs include P-3 agonists, CB-1antagonists, appetite suppressants, such as, for example, sibutramine(Meridia), and lipase inhibitors, such as, for example, orlistat(Xenical). The subject copolymers may also be used in methods of theinvention in combination with drugs commonly used to treat lipiddisorders in diabetic patients. Such drugs include, but are not limitedto, HMG-CoA reductase inhibitors, nicotinic acid, bile acidsequestrants, and fibric acid derivatives. Polypeptides of the inventionmay also be used in combination with anti-hypertensive drugs, such as,for example, β-blockers, cathepsin S inhibitors and ACE inhibitors.Examples of β-blockers are: acebutolol, bisoprolol, esmolol, propanolol,atenolol, labetalol, carvedilol, and metoprolol. Examples of ACEinhibitors are: captopril, enalapril, lisinopril, benazepril,fosinopril, ramipril, quinapril, perindopril, trandolapril, andmoexipril.

The invention further provides a kit comprising (i) a compositioncomprising a random copolymer and (ii) instructions for administeringthe composition to a subject in need thereof at intervals greater than24 hours, more preferably greater than 36 hours, for the treatment of adisease, such as an autoimmune disease. In one embodiment, theautoimmune disorder is multiple sclerosis. In a preferred embodiment,the random copolymer is Copolymer 1. In another preferred embodiment,the random copolymer is formulated in dosages for administration ofgreater than about 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96,102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180,186, 192, 198, 204, 210, 216, 222, 228, 234, or 240 hours, or anyintervening interval thereof. In another embodiment of the kitsdescribed herein, the instructions indicate that the random polymer isto be administered every about 24, 30, 36, 42, 48, 54, 60, 66, 72, 78,84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168,174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, or 240 hours, orany interval in between. Kits may comprise additional components, suchas packaging and one or more apparatuses for the administration of thecopolymer, such as a hypodermic syringe.

In a specific embodiment, the autoimmune disease is selected from thegroup consisting of multiple sclerosis, type-I diabetes, Hashimoto'sthyroiditis, Crohn's disease, rheumatoid arthritis, systemic lupuserythematosus (SLE), gastritis, autoimmune hepatitis, hemolytic anemia,autoimmune hemophilia, autoimmune lymphoproliferative syndrome (ALPS),autoimmune uveoretinitis, glomerulonephritis, Guillain-Barré syndrome,psoriasis, myasthenia gravis, autoimmune encephalomyelitis,Goodpasture's syndrome, Grave's disease, paraneoplastic pemphigus,autoimmune thrombocytopenic purpura, scleroderma with anti-collagenantibodies, mixed connective tissue disease, pernicious anemia,polymyositis, idiopathic Addison's disease, autoimmune-associatedinfertility, bullous pemphigoid, Sjogren's syndrome, idiopathic myxedemaand colitis.

VI. Methods of Treatment

One aspect of the present invention provides novel methods to treat asubject afflicted with or suspected of being afflicted with a disease,such as an autoimmune disease, by administering one or more randomcopolymers to the subject in a therapeutically effective amount. Inparticular, subcutaneous administration of a pharmaceutical compositioncomprising a random copolymer composition is contemplated as a preferredembodiment of the invention. Subcutaneous injection induces more desiredimmune responses biased for TH2 response, which is the basis for thetolerance for certain antigens.

In general, the methods of treatment of the present invention, which isimmunomodulation of the subject in need of such treatment, can bedifferentiated from vaccination. Successful vaccination is dependent onthe immunogenicity of the vaccine being administered, which increasesthe titer of antibodies directly reactive to the antigens in thevaccine. In contrast, the random copolymers of the present invention areeffective in treating diseases without inducing a high titer ofantibodies against the copolymers themselves. As demonstrated by theExamples below, the effectiveness of the methods of the presentinvention does not depend on the antibody production against thecopolymers, and therefore is fundamentally different from vaccination.Unlike vaccination, random copolymers of the present invention,administered by the methods of the invention, induces tolerance towardthe disease-related antigens, and more specifically, induces peripheraltolerance. Peripheral tolerance, in contract to central tolerance, hasthe advantage of being safer as a modulatory phenomenon. Accordingly,one aspect of the present invention is embodied in a method ofadministration of a composition comprising a random copolymer of theinvention so as to induce peripheral tolerance toward the randomcopolymer and the disease related antigens.

In general, an embodiment of the invention is to administer a suitabledose of a therapeutic copolymer composition that will be the lowesteffective dose to produce a therapeutic effect, for example, mitigatingsymptoms. The therapeutic copolymers are preferably administered at adose per subject, which corresponds to a dose per day of at least about2 mg, at least about 5 mg, at least about 10 mg, or at least about 20 mgas appropriate minimal starting dosages, or about x mg, wherein x is aninteger between 1 and 20. In one embodiment of the methods describedherein, a dose of about 0.01 to about 500 mg/kg can be administered. Ingeneral, the effective dosage of the compound of the present inventionis about 50 to about 400 micrograms of the compound per kilogram of thesubject per day. In one specific embodiment, the equivalent dosage perday, regardless of the frequency with which the doses are administered,is from about 5 to 100, or more preferably, from about 10 to 40, or morepreferably about 20 mg/day. In another specific embodiment, eachindividual dosage in the treatment regimen is from about 5 to 100, ormore preferably from about 10 to 40, or more preferably about 20mg/dose.

However, it is understood by one skilled in the art that the dose of thecomposition of the invention will vary depending on the subject and uponthe particular route of administration used. It is routine in the art toadjust the dosage to suit the individual subjects. Additionally, theeffective amount may be based upon, among other things, the size of thecompound, the biodegradability of the compound, the bioactivity of thecompound and the bioavailability of the compound. If the compound doesnot degrade quickly, is bioavailable and highly active, a smaller amountwill be required to be effective. The actual dosage suitable for asubject can easily be determined as a routine practice by one skilled inthe art, for example a physician or a veterinarian given a generalstarting point. For example, the physician or veterinarian could startdoses of the compound of the invention employed in the pharmaceuticalcomposition at a level lower than that required in order to achieve thedesired therapeutic effect, and increase the dosage with time until thedesired effect is achieved. A physician or veterinarian may also referto the recommendations for the administration of Copaxone™ as a generalstarting point.

In the context of the invention, the term “treatment regimen” is meantto encompass therapeutic, palliative and prophylactic modalities ofadministration of one or more compositions comprising one or more randomcopolymers. A particular treatment regimen may last for a period of timewhich will vary depending upon the nature of the particular disease ordisorder, its severity and the overall condition of the patient, and mayextend from once daily, or more preferably once every 36 hours or 48hours or longer, to once every month or several months. Followingtreatment, the patient is monitored for changes in his/her condition andfor alleviation of the symptoms of the disorder or disease state. Thedosage of the oligonucleotide may either be increased in the event thepatient does not respond significantly to current dosage levels, or thedose may be decreased if an alleviation of the symptoms of the disorderor disease state is observed, or if the disorder or disease state hasbeen ablated, or if an unacceptable side effects are seen with thestarting dosage.

In one embodiment, a therapeutically effective amount of the randomcopolymer is administered to the subject in a treatment regimencomprising intervals of at least 36 hours, or more preferably 48 hours,between dosages. In another embodiment, the random copolymer isadministered at intervals of at least 54, 60, 66, 72, 78, 84, 90, 96,102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180,186, 192, 198, 204, 210, 216, 222, 228, 234, or 240 hours, or theequivalent amount of days. In some embodiments, the agent isadministered every other day, while in other embodiments it isadministered weekly. If two copolymers are administered to the subject,such copolymers may be administered at the same time, such assimultaneously, or essentially at the same time, such as in succession.Alternatively, their administration may be staggered. For example, twocopolymers which are each administered every 48 hours may both beadministered on the same days, or one may be administered one day andthe other on the next day and so on in an alternating fashion.

As shown by the Examples below, treatment regimens with longer dosingintervals, consequently often with lower total exposure of copolymers,induce lower titers of antibodies against copolymers themselves, whilestill inducing desired protective effects. Such reduction ofneutralizing antibodies are desirable because it is considered likely tohelp random copolymer compositions to retain its effectiveness withoutbeing neutralized, and it is associated with reduced risk ofanaphylactic shocks, providing safer treatments of diseases. Longerinterval regimens are also desirable because they strengthen the biasfor TH2 responses, which is considered to be the mode of action for therandom copolymer therapies.

In other embodiments, the random copolymer is administered in atreatment regimen which comprises at least one uneven time interval,wherein at least one of the time intervals is at least 24, 30, 36, 42,48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138,144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222,228, 234, or 240 hours, or the equivalent amount of days.

In one embodiment, the polymer is administered to be subject at leastthree times during a treatment regimen, such that there are at least twotime intervals between administrations. These intervals may be denotedI₁ and I₂. If the polymer is administered four times, then there wouldbe an additional interval between the third and fourth administrations,I₃, such that the number of intervals for a given number “n” ofadministrations is n−1. Accordingly, in one embodiment, at least one ofthe time intervals between administrations is greater than about 24, 30,36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126,132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210,216, 222, 228, 234, or 240 hours. In another embodiment, at least 1%,2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or95% of the total number n−1 of time intervals are at least about 24, 30,36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126,132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210,216, 222, 228, 234, or 240 hours.

In yet another embodiment, the average time interval betweenadministrations ((I₁+I₂+ . . . +I_(n-1))/n−1) is at least 24, 30, 36,42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132,138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216,222, 228, 234, or 240 hours, or at least two weeks.

In another embodiment, the dosage regimen consists of two or moredifferent interval sets. For example, a first part of the dosage regimenis administered to a subject daily, every other day, or every third day,for example, at about 22 mg copolymer/m² body surface area of thesubject, wherein the subject is a human. In some embodiment of theinvention, the dosing regimen starts with dosing the subject every otherday, every third day, weekly, biweekly, or monthly. The dosage foradministration every other day or every third day may be up to about 65mg/m² and 110 mg/m² respectively. For a dosing regimen comprising dosingof the random copolymer every week, the dose comprises up to about 500mg/m², and for a dosing regimen comprising dosing of the randomcopolymer every two weeks or every month, up to 1.5 g/m² may beadministered. The first part of the dosing regimen may be administeredfor up to 30 days, for example, 7, 14, 21, or 30 days. A subsequentsecond part of the dosing regimen with a different, longer intervaladministration with usually lower exposure (step-down dosage),administered weekly, every 14 days, or monthly may optionally follow,for example, at 500 mg/m² body surface area weekly, up to maximum ofabout 1.5 g/m² body surface area, continuing for 4 weeks up to twoyears, for example, 4, 6, 8, 12, 16, 26, 32, 40, 52, 63, 68, 78, or 104weeks. Alternatively, if the disease goes into remission or generallyimproves, the dosage may be maintained or kept at lower than maximumamount, for example, at 140 mg/m² body surface area weekly. If, duringthe step-down dosage regimen, the disease condition relapses, the firstdosage regimen may be resumed until effect is seen, and the seconddosing regimen may be implemented. This cycle may be repeated multipletimes as necessary.

More specifically, one aspect of the invention is treatment of diseasestreatable with a random copolymer. One embodiment of the invention is amethod for treating diseases treatable with random copolymer of thecomposition YFAK (L-tyrosine, L-phenylalanine, L-alanine and L-lysine)in a molar input ratio of about 1.0:1.0:10.0:6.0 respectively,synthesized by solid phase chemistry, wherein the copolymer has a lengthof 52 amino acids, by administering said random copolymer to a humansubject in need of treatment a first part of a dosing regimen comprisinga dose of about 22 mg/m² body surface area daily. In some embodiment ofthe invention, the dosing regimen starts with dosing the subject everyother day, every third day, weekly, biweekly, or monthly. The dosage foradministration every other day or every third day may be up to about 65mg/m² and 110 mg/m² respectively. For a dosing regimen comprising dosingof the random copolymer every week, the dose comprises up to about 500mg/m², and for a dosing regimen comprising dosing of the randomcopolymer every two weeks or every month, up to 1.5 g/m² may beadministered. The first part of the dosing regimen may be administeredfor up to 30 days, for example, 7, 14, 21, or 30 days. A subsequentsecond part of the dosing regimen with a different, longer intervaladministration with usually lower exposure (step-down dosage),administered weekly, every 14 days, or monthly may optionally follow,for example, at 500 mg/m² body surface area weekly, up to maximum ofabout 1.5 g/m² body surface area, continuing for 4 weeks up to twoyears, for example, 4, 6, 8, 12, 16, 26, 32, 40, 52, 63, 68, 78, or 104weeks. Alternatively, if the disease goes into remission or generallyimproves, the dosage may be maintained or kept at lower than maximumamount, for example, at 140 mg/m² body surface area weekly. If, duringthe step-down dosage regimen, the disease condition relapses, the firstdosage regimen may be resumed until effect is seen, and the seconddosing regimen may be implemented. This cycle may be repeated multipletimes as necessary.

In another embodiment of the invention, the method is for treatingdiseases treatable with Copolymer 1 (YEAK) of about 52 amino acidlength, having a molar input ratio of about 1.0:2.0:6.0:5.0, andsynthesized by solid phase chemistry. The dosage regimen is similar tothat described for YFAK above.

Another embodiment of the invention is a method of treating diseasestreatable with a random copolymer of the composition YFAK (L-tyrosine,L-phenylalanine, L-alanine and L-lysine) in an output average molarratio of about 1.0:1.2:18.0:6.0 respectively, synthesized by solid phasechemistry, wherein the copolymer has a length of 52 amino acids, andwherein residues 1-10 of the copolymer sequence has a molar output ratioof about 1.0:1.2:16:6, residues 11-30 have a molar output ratio of about1.0:1.2:18:6, and residues 31-52 have a molar output ratio of about1.0:1.2:20:6 by administering said random copolymer to a human subjectin need of treatment a dose of about 22 mg/m² body surface area daily,or with longer intervals such as every other day, every third day,weekly, biweekly, or monthly, as described above. In another embodimentof the invention, the method is for treating diseases treatable withCopolymer 1 (YEAK) of about 52 amino acid length, having a molar inputratio of about 1.0:2.0:6.0:5.0, and synthesized by solid phasechemistry, wherein residues 1-10 of the copolymer sequence has a molaroutput ratio of about 1.0:2.0:5.5:5.0, residues 11-30 have a molaroutput ratio of about 1.0:2.0:6.0:5.0, and residues 31-52 have a molaroutput ratio of about 1.0:2.0:6.5:5.0. The dosage regimen is similar tothat described above, and may optionally include the step-down sage. If,during the step-down dosage regimen, the disease condition relapses, thefirst dosage regimen may be resumed until effect is seen, and the seconddosing regimen may be implemented. This cycle may be repeated multipletimes as necessary.

Another aspect of the invention is embodied as a means for amelioratingdiseases treatable with a random copolymer comprising the compositionYFAK (L-tyrosine, L-phenylalanine, L-alanine and L-lysine) in an molarinput ratio of about 1.0:1.0:XA:6.0, wherein XA is a number greater than5.0 and less than 15.0 respectively by administering to a subject a doseeffective in ameliorating said diseases. More specifically, oneembodiment of the invention is a means for ameliorating diseasestreatable with a random copolymer of the composition YFAK (L-tyrosine,L-phenylalanine, L-alanine and L-lysine) in an output average molarratio of about 1.0:1.2:18.0:6.0 respectively, synthesized by solid phasechemistry, wherein the copolymer has a length of 52 amino acids, andwherein residues 1-10 of the copolymer sequence has a ratio of about1.0:1.2:16:6, residues 11-30 have a ratio of about 1.0:1.2:18:6, andresidues 31-52 have a ratio of about 1.0:1.2:20:6. In another embodimentof the invention, the method is for treating diseases treatable withCopolymer 1 (YEAK) of about 52 amino acid length, having a molar inputratio of about 1.0:2.0:6.0:5.0, and synthesized by solid phasechemistry, wherein residues 1-10 of the copolymer sequence has a molaroutput ratio of about 1.0:2.0:5.5:5.0, residues 11-30 have a molaroutput ratio of about 1.0:2.0:6.0:5.0, and residues 31-52 have a molaroutput ratio of about 1.0:2.0:6.5:5.0. The subject is treated followinga dosage regimen at about 22 mg copolymer/m² body surface area of thesubject, wherein the subject is a human. In some embodiment of theinvention, the dosing regimen starts with dosing the subject every otherday, every third day, weekly, biweekly, or monthly. The dosage foradministration every other day or every third day may be up to about 65mg/m² and 110 mg/m² respectively. For a dosing regimen comprising dosingof the random copolymer every week, the dose comprises up to about 500mg/m², and for a dosing regimen comprising dosing of the randomcopolymer every two weeks or every month, up to 1.5 g/m² may beadministered. The first part of the dosing regimen may be administeredfor up to 30 days, for example, 7, 14, 21, or 30 days. A subsequentsecond part of the dosing regimen with a different, longer intervaladministration with usually lower exposure (step-down dosage),administered weekly, every 14 days, or monthly may optionally follow,for example, at 500 mg/m² body surface area weekly, up to maximum ofabout 1.5 g/m² body surface area, continuing for 4 weeks up to twoyears, for example, 4, 6, 8, 12, 16, 26, 32, 40, 52, 63, 68, 78, or 104weeks. Alternatively, if the disease goes into remission or generallyimproves, the dosage may be maintained or kept at lower than maximumamount, for example, at 140 mg/m² body surface area weekly. If, duringthe step-down dosage regimen, the disease condition relapses, the firstdosage regimen may be resumed until effect is seen, and the seconddosing regimen may be implemented. This cycle may be repeated multipletimes as necessary.

An aspect of the invention is a means for ameliorating unwanted immuneresponses by administering to a subject a dose effective in amelioratingsaid diseases with a random copolymer comprising the composition YFAK(L-tyrosine, L-phenylalanine, L-alanine and L-lysine) in an molar inputratio of about 1.0:1.0:10.0:6.0 respectively. In another embodiment ofthe invention, the method is for treating diseases treatable withCopolymer 1 (YEAK) of about 52 amino acid length, having a molar inputratio of about 1.0:2.0:6.0:5.0, and synthesized by solid phasechemistry. The dosage regimen is similar to that described for YFAKherein. For both types of random copolymers, an exemplary means is byadministering to a human subject a daily dose of about 22 mg randomcopolymer/m² body surface area. In some embodiment of the invention, thedosing regimen starts with dosing the subject every other day, everythird day, weekly, biweekly, or monthly. The dosage for administrationevery other day or every third day may be up to about 65 mg/m² and 110mg/m² respectively. For a dosing regimen comprising dosing of the randomcopolymer every week, the dose comprises up to about 500 mg/m², and fora dosing regimen comprising dosing of the random copolymer every twoweeks or every month, up to 1.5 g/m² may be administered. The first partof the dosing regimen may be administered for up to 30 days, forexample, 7, 14, 21, or 30 days. A subsequent second part of the dosingregimen with a different, longer interval administration with usuallylower exposure (step-down dosage), administered weekly, every 14 days,or monthly may optionally follow, for example, at 500 mg/m² body surfacearea weekly, up to maximum of about 1.5 g/m² body surface area,continuing for 4 weeks up to two years, for example, 4, 6, 8, 12, 16,26, 32, 40, 52, 63, 68, 78, or 104 weeks. Alternatively, if the diseasegoes into remission or generally improves, the dosage may be maintainedor kept at lower than maximum amount, for example, at 140 mg/m² bodysurface area weekly. If, during the step-down dosage regimen, thedisease condition relapses, the first dosage regimen may be resumeduntil effect is seen, and the second dosing regimen may be implemented.This cycle may be repeated multiple times as necessary.

Yet another embodiment of the invention is a means for amelioratingunwanted immune responses with a random copolymer of the compositionYFAK (L-tyrosine, L-phenylalanine, L-alanine and L-lysine) in an outputaverage molar ratio of about 1.0:1.2:18.0:6.0 respectively, synthesizedby solid phase chemistry, wherein the copolymer has a length of 52 aminoacids, and wherein residues 1-10 of the copolymer sequence has a ratioof about 1.0:1.2:16:6, residues 11-30 have a ratio of about1.0:1.2:18:6, and residues 31-52 have a ratio of about 1.0:1.2:20:6. Inanother embodiment of the invention, the method is for treating diseasestreatable with Copolymer 1 (YEAK) of about 52 amino acid length, havinga molar input ratio of about 1.0:2.0:6.0:5.0, and synthesized by solidphase chemistry, wherein residues 1-10 of the copolymer sequence has amolar output ratio of about 1.0:2.0:5.5:5.0, residues 11-30 have a molaroutput ratio of about 1.0:2.0:6.0:5.0, and residues 31-52 have a molaroutput ratio of about 1.0:2.0:6.5:5.0. The dosage regimen is similar tothat described for YFAK above. For both types of random copolymers, suchmethod may be carried out by administering to a human subject a dailydose of about 22 mg random copolymer/m² body surface area. Dosingregimens may be similar to those described above, tailored to thesubject's needs. Alternatively, the random copolymer may be administeredto a human subject at a maximum daily dose of about 80 mg.

Another aspect of the invention is a method for ameliorating unwantedimmune responses having a TH1 phenotype with a random copolymer of thecomposition YFAK (L-tyrosine, L-phenylalanine, L-alanine and L-lysine)in an output average molar ratio of about 1.0:1.2:18.0:6.0 respectively,synthesized by solid phase chemistry, wherein the copolymer has a lengthof 52 amino acids, and wherein residues 1-10 of the copolymer sequencehas a ratio of about 1.0:1.2:16:6, residues 11-30 have a ratio of about1.0:1.2:18:6, and residues 31-52 have a ratio of about 1.0:1.2:20:6. Inanother embodiment of the invention, the method is for treating diseasestreatable with Copolymer 1 (YEAK) of about 52 amino acid length, havinga molar input ratio of about 1.0:2.0:6.0:5.0, and synthesized by solidphase chemistry, wherein residues 1-10 of the copolymer sequence has amolar output ratio of about 1.0:2.0:5.5:5.0, residues 11-30 have a molaroutput ratio of about 1.0:2.0:6.0:5.0, and residues 31-52 have a molaroutput ratio of about 1.0:2.0:6.5:5.0. For both types of randomcopolymers, the dosage regimen may be determined, tailored to thesubject's needs, and can be similar to that described above.

Yet another aspect of the invention is a means for amelioratingautoimmune reactions in a subject with a random copolymer of thecomposition YFAK (L-tyrosine, L-phenylalanine, L-alanine and L-lysine)in an output average molar ratio of about 1.0:1.2:18.0:6.0 respectively,synthesized by solid phase chemistry, wherein the copolymer has a lengthof 52 amino acids, and wherein residues 1-10 of the copolymer sequencehas a ratio of about 1.0:1.2:16:6, residues 11-30 have a ratio of about1.0:1.2:18:6, and residues 31-52 have a ratio of about 1.0:1.2:20:6. Inanother embodiment of the invention, the method is for treating diseasestreatable with Copolymer 1 (YEAK) of about 52 amino acid length, havinga molar input ratio of about 1.0:2.0:6.0:5.0, and synthesized by solidphase chemistry, wherein residues 1-10 of the copolymer sequence has amolar output ratio of about 1.0:2.0:5.5:5.0, residues 11-30 have a molaroutput ratio of about 1.0:2.0:6.0:5.0, and residues 31-52 have a molaroutput ratio of about 1.0:2.0:6.5:5.0. For both types of randomcopolymers, dosing regimens may be similar to those described above,tailored to the subject's needs.

Any of the methods and means may be practiced using compositions andformulations described in this application.

In other embodiments of the invention, any of the methods of theinvention may be practiced using sustained release formulationcomprising a random copolymer. When administering a random copolymer ofthe invention using a sustained release formula, the overall exposure tothe copolymer is generally lower than in bolus administration. Forexample, a first part of the dosage regimen is administered to a subjectdaily, every other day, or every third day, for example, at about 22 mgcopolymer/m² body surface area of the subject, wherein the subject is ahuman. In some embodiment of the invention, the dosing regimen usessustained release formula, dosing the subject every other day, everythird day, weekly, biweekly, or monthly so that the copolymer isreleased during the interval. The dosage for administration every otherday or every third day may be up to about 35 mg/m² and 65 mg/m²respectively. For a dosing regimen comprising dosing of the randomcopolymer every week, the dose comprises up to about 140 mg/m², and fora dosing regimen comprising dosing of the random copolymer every twoweeks or every month, up to 750 mg/m² may be administered. The firstpart of the dosing regimen may be administered for up to 30 days, forexample, 7, 14, 21, or 30 days. A subsequent second part of the dosingregimen with a different, longer interval administration with usuallylower exposure (step-down dosage), administered weekly, every 14 days,or monthly may optionally follow, for example, at 140 mg/m² body surfacearea weekly, up to maximum of about 1.5 g/m² body surface area,continuing for 4 weeks up to two years, for example, 4, 6, 8, 12, 16,26, 32, 40, 52, 63, 68, 78, or 104 weeks. Alternatively, if the diseasegoes into remission or generally improves, the dosage may be maintainedor kept at lower than maximum amount, for example, at 140 mg/m² bodysurface area weekly. If, during the step-down dosage regimen, thedisease condition relapses, the first dosage regimen may be resumeduntil effect is seen, and the second dosing regimen may be implemented.This cycle may be repeated multiple times as necessary.

Another aspect of the invention is a means for treating a subjectafflicted with or showing the symptoms of multiple sclerosis (MS) with arandom copolymer of the composition YFAK (L-tyrosine, L-phenylalanine,L-alanine and L-lysine) in an output average molar ratio of about1.0:1.2:18.0:6.0 respectively, synthesized by solid phase chemistry,wherein the copolymer has a length of 52 amino acids, and whereinresidues 1-10 of the copolymer sequence have a ratio of about1.0:1.2:16:6, residues 11-30 have a ratio of about 1.0:1.2:18:6, andresidues 31-52 have a ratio of about 1.0:1.2:20:6. In another embodimentof the invention, the method is for treating diseases treatable withCopolymer 1 (YEAK) of about 52 amino acid length, having a molar inputratio of about 1.0:2.0:6.0:5.0, and synthesized by solid phasechemistry, wherein residues 1-10 of the copolymer sequence has a molaroutput ratio of about 1.0:2.0:5.5:5.0, residues 11-30 have a molaroutput ratio of about 1.0:2.0:6.0:5.0, and residues 31-52 have a molaroutput ratio of about 1.0:2.0:6.5:5.0. Such copolymers may beadministered to treat a subject afflicted with or showing the symptomsof MS with a maximum dose of 500 mg a random copolymer of abovedescribed random copolymer. The random copolymer may be delivered in asustained release formulation.

Means for treating a subject suffering from multiple sclerosis with amaximum dose of 500 mg delivered in a sustained release formulation arandom copolymer of the composition YFAK (L-tyrosine, L-phenylalanine,L-alanine and L-lysine) in an output average molar ratio of about1.0:1.2:18.0:6.0 respectively, synthesized by solid phase chemistry,wherein the copolymer has a length of 52 amino acids, and whereinresidues 1-10 of the copolymer sequence have a ratio of about1.0:1.2:16:6, residues 11-30 have a ratio of about 1.0:1.2:18:6, andresidues 31-52 have a ratio of about 1.0:1.2:20:6. In another embodimentof the invention, the method is for treating diseases treatable withCopolymer 1 (YEAK) of about 52 amino acid length, having a molar inputratio of about 1.0:2.0:6.0:5.0, and synthesized by solid phasechemistry, wherein residues 1-10 of the copolymer sequence has a molaroutput ratio of about 1.0:2.0:5.5:5.0, residues 11-30 have a molaroutput ratio of about 1.0:2.0:6.0:5.0, and residues 31-52 have a molaroutput ratio of about 1.0:2.0:6.5:5.0. Alternatively, the subject may betreated with a weekly maximum dose of 500 mg of the random copolymer,delivered in sustained release formulation.

In any of the exemplary embodiments described above, the volume of theeach dosage form is preferably 0.1 ml to 5 ml.

In one embodiment of the methods described herein, the route ofadministration can be oral, intraperitoneal, transdermal, subcutaneous,by intravenous or intramuscular injection, by inhalation, topical,intralesional, infusion; liposome-mediated delivery; topical,intrathecal, gingival pocket, rectal, intravaginal, intrabronchial,nasal, transmucosal, intestinal, ocular or otic delivery, or any othermethods known in the art as one skilled in the art may easily perceive.Other embodiments of the compositions of the invention incorporateparticulate forms protective coatings, protease inhibitors or permeationenhancers for various routes of administration, including parenteral,pulmonary, nasal and oral. Administration can be systemic or local. In apreferred embodiment, the random copolymer is administeredsubcutaneously.

An embodiment of the methods of present invention relates to theadministration of the copolymers of the present invention in a sustainedrelease form. Such method comprises applying a sustained-releasetransdermal patch or implanting a sustained-release capsule or a coatedimplantable medical device so that a therapeutically effective dose ofthe copolymer of the present invention is delivered at defined timeintervals to a subject of such a method. The compounds and/or agents ofthe subject invention may be delivered via a capsule which allowsregulated-release of the random copolymer over a period of time.Controlled or sustained-release compositions include formulation inlipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended bythe invention are particulate compositions coated with polymers (e.g.,poloxamers or poloxamines). In certain embodiments, a source of acopolymer is stereotactically provided within or proximate to the areaof autoimmune attack, for example, near the pancreas for the treatmentof IDDM.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pre-gelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulfate). The tablets may be coated by methodswell-known in the art.

When Copolymer 1 or other random copolymer is introduced orally, it maybe mixed with other food forms and consumed in solid, semi-solid,suspension, or emulsion form; and it may be mixed with pharmaceuticallyacceptable carriers, including water, suspending agents, emulsifyingagents, flavor enhancers, and the like. In one embodiment, the oralcomposition is enterically-coated. Use of enteric coatings is well knownin the art. For example, Lehman (1971) teaches enteric coatings such asEudragit S and Eudragit L. The Handbook of Pharmaceutical Excipients,2^(nd) Ed., also teaches Eudragit S and Eudragit L applications. OneEudragit which may be used in the present invention is L30D55.Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner. The compositions may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen free water, before use.

The compositions may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. Foradministration by inhalation, the compositions for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin, for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

In a preferred embodiment, compositions comprising Copolymer 1 oranother random copolymer are formulated in accordance with routineprocedures as pharmaceutical compositions adapted for intravenousadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic such as lignocaine to ease pain at the site of theinjection. Generally, the ingredients are supplied either separately ormixed together. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline, with the intervals betweenadministrations being greater than 24 hours, 32 hours, or morepreferably greater than 36 or 48 hours. Where the composition isadministered by injection, an ampoule of sterile water or saline forinjection can be provided so that the ingredients may be mixed prior toadministration.

In certain embodiments, the methods described herein allow continuoustreatment of autoimmune diseases by a sustained-release carrier such astransdermal patches, implantable medical devices coated withsustained-release formulations, or implantable or injectablepharmaceutical formulation suitable for sustained-release of the activecomponents. In such embodiments, the intervals between administrationsare preferably greater than 24 hours, 32 hours, or more preferablygreater than 36 or 48 hours. For instance, an implantable device or asustained released formulation which releases the copolymer over a 2 dayperiod may the implanted every four days into the patient, such that theinterval during which no copolymer is administered to the subject is 2days. In related embodiments, the such interval where during which noadministration occurs is at least 24+x hours, wherein x represents anypositive integer.

In another embodiment, the random copolymers are formulated to have atherapeutic affect when administered to a subject in need thereof attime intervals of at least 24 hours. In a specific embodiment, therandom copolymers are formulated for a long-lasting therapeutic affectsuch that a therapeutic effect in treating the disease is observed whenthe random copolymers are administered to the subject at time intervalsof at least 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102,108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186,192, 198, 204, 210, 216, 222, 228, 234, or 240 hours betweenadministrations.

Another embodiment of the present invention is a method forprophylactically treating a subject at risk of developing e.g., anautoimmune disease by administering a random copolymer. A subject atrisk is identified by, for example, determining the geneticsusceptibility to an autoimmune disease by testing for alleles of HLAthat are associated with such autoimmune disease, and/or based onfamilial history, or other genetic markers that correlate with suchautoimmune disease. Such prophylactic treatment may additionallycomprise a second copolymer that binds to a second HLA moleculeassociated with the autoimmune disease to be treated. The second HLAmolecule may be a HLA-DQ or HLA-DR molecule. Preferably, the autoimmunedisease to be prophylactically treated is IDDM or celiac disease.

In other embodiments of the methods described herein, additionaltherapeutically active agents are administered to the subject. In oneembodiment, compositions comprising additional therapeutic agents(s) areadministered to the subject as separate compositions from thosecomprising the random polymer. For example, a subject may beadministered a composition comprising a random copolymer subcutaneouslywhile a composition comprising another therapeutic agent may beadministered orally. The additional therapeutically active agents maytreat the same disease as the random copolymer, a related disease, ormay be intended to treat an undesirable side effect of administration ofthe copolymer, such as to reduce swelling at a site of intradermalinjection.

Additional therapeutically active agents which may be administered tothe subject include copolymers which bind to a second HLA moleculeassociated with the disease, such as Copaxone™; an antibody, an enzymeinhibitor, an antibacterial agent, an antiviral agent, a steroid, anonsteroidal anti-inflammatory agent, an antimetabolite, a cytokine, ora soluble cytokine receptor. The second HLA molecule may be an HLA-DQmolecule or an HLA-DR molecule. The enzyme inhibitor may be a proteaseinhibitor or a cyclooxygenase inhibitor.

In a further embodiment, the copolymers of the present invention areadministered to a patient with an autoimmune disease following ananti-lymphocyte therapy (e.g., anti-T cell or anti-B cell). In oneembodiment, anti-T cell therapies may use antibodies, such asCampath-1H® (alemtuzumab; anti-CD52), OKT3 (anti-CD3), thymoglobulin(anti-thymocytic globulins), or anti-IL2R antibodies (e.g., daclizumaband basiliximab). Alternatively, anti-T cell therapies may usechemotherapy agents such as fludarabine, external-beam radiation therapy(XRT), and cyclophosphamide. In another embodiment, the copolymers ofthe present invention may be combined with an anti-B cell therapy fortreating an autoimmune disease, such as agents that react with CD20 suchas the antibody Rituxan (Rituximab). The dosage of the above treatmentsto be administered to a patient will vary with the precise nature of thecondition being treated and the recipient of the treatment. The scalingof dosages for human administration can be performed according toart-accepted practices. For example, the dose for Campath-1H® willgenerally be in the range 1 to about 100 mg for an adult patient,usually administered daily for a period between 1 and 30 days. Thepreferred daily dose is 1 to 10 mg per day although in some instanceslarger doses of up to 40 mg per day may be used (see, e.g., U.S. Pat.No. 6,120,766). Although not wishing to be bound by any particularmechanism or theory, it is believed that such combination therapy canenhance the therapeutic efficacy without any potential long-termtoxicity. To illustrate, Campath-1H® is introduced in a patient forinitial induction immunosuppression. Then, the patient is administered acopolymer of the present invention in the absence of Campath-1H®.

In other embodiments, a copolymer of the present invention can beadministered with an anti-lymphocyte agent (e.g., anti-T cell or anti-Bcell) either in the same formulation or in separate formulations, toenhance treatment. For example, a copolymer and an anti-lymphocyte agentcan be administered at the same time (simultaneously) or at separatetimes (sequentially), provided that they are administered in such amanner and sufficiently close in time to have the desired effect.

The additional agent may be added as a part of the pharmaceuticalcomposition, or may be administered concomitantly or within a timeperiod when the physiological effect of the additional agent overlapswith the physiological effect of the copolymer of the present invention.More particularly, an additional agent may be administered concomitantlyor one week, several days, 24 hours, 8 hours, or immediately before theadministration of the copolymer. Alternatively, an additional agent maybe administered one week, several days, 24 hours, 8 hours, orimmediately after the administration of the copolymer.

An improvement in the symptoms of a subject afflicted with multiplesclerosis (MS) as a result of administration of the random copolymer maybe noted by a decrease in frequency of recurrences of episodes of MS, bydecrease in severity of symptoms, and by elimination of recurrentepisodes for a period of time after the start of administration. Atherapeutically effective dosage preferably reduces symptoms andfrequency of recurrences by at least about 20%, for example, by at leastabout 40%, by at least about 60%, and by at least about 80%, or by about100% elimination of one or more symptoms, or elimination of recurrencesof the autoimmune disease, relative to untreated subjects. The period oftime can be at least about one month, at least about six months, or atleast about one year.

An improvement in the symptoms of a subject afflicted with arthritis orany other autoimmune disorder which results in inflammation of thejoints may be noted by a reduction in edema of one or more joints, by areduction in inflammation in one or more joints, or by an increase inmobility in one or more joints. A therapeutically effective dosagepreferably reduces joint inflammation and edema and improves mobility byat least about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and even still more preferably by atleast about 80%, relative to untreated subjects.

The contents of any patents, patent applications, patent publications,or scientific articles referenced anywhere in this application areherein incorporated in their entirety.

The practice of the present invention will employ, where appropriate andunless otherwise indicated, conventional techniques of cell biology,cell culture, molecular biology, transgenic biology, microbiology,virology, recombinant DNA, and immunology, which are within the skill ofthe art. Such techniques are described in the literature. See, forexample, Molecular Cloning: A Laboratory Manual, 3rd Ed., ed. bySambrook and Russell (Cold Spring Harbor Laboratory Press: 2001); thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); UsingAntibodies, Second Edition by Harlow and Lane, Cold Spring Harbor Press,New York, 1999; Current Protocols in Cell Biology, ed. by Bonifacino,Dasso, Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons,Inc., New York, 1999; and PCR Protocols, ed. by Bartlett et al., HumanaPress, 2003.

VII. EXAMPLES Example 1 Production of Antibodies Against RandomCopolymers and a Disease-Associated Antigen Peptide

PLP(139-151) peptide is the major immunogenic determinant recognized byCD4⁺ TH1 cells which in turn drive EAE development in SJL mice. Wheninjected with pertussis toxin, PLP (139-151) peptide causes MS-likesymptoms in the SJL mice. In the absence of the pertussis toxin,injected animals develop only mild and transient disease. The ability ofrandom copolymer compositions to protect the animals from the effect ofPLP injection was evaluated in the course of daily and weekly dosing ofthe animals after their exposure to PLP (139-151) peptide. Antibodyisotypes were also examined. CD4 T cells can be divided into at leasttwo different subsets depending on the pattern of their cytokineproduction. TH1 cells preferentially produce IL-2 and IFN-γ, activatemacrophages, and stimulate production of the Ig subclasses IgG2a andIgG3 in mice and IgG1 and IgG3 in humans. In contrast, the signaturecytokines of TH2 cells are IL-4, IL-5, and IL-13, which provide potent Bcell help and induce isotype switching to IgE and IgG1 in mice or toIgE, IgG2, and IgG4 in humans. Therefore, mouse IgG1 and IgG2b,generally associated with TH2 response, and mouse IgG2a, markers of TH1immunity, were measured.

Mice (SJL, female) were immunized on day 1 with 100 μg of PLP(139-151)peptide in Complete Freund's adjuvant. The same day, the animalsreceived an intravenous injection of 200 ng of pertussis toxin. On day3, the same IV injection was repeated. Treatment with Copaxone™ (YEAK)or Co-14 (YFAK), 7.5 mg/kg, daily and weekly was started on day 6 andcontinued daily until day 36. On day 37, individual sera were collectedand antibody response against PLP (139-151) peptide, Co-14 (YFAK), andCopaxone™ were measured using standard ELISA with anti-mouse total Ig,IgG1, IgG2a or IgG2b as secondary antibody.

During the course of the experiment, disease severity was measured usinga standard scoring system between 0 (no disease) and 5 (moribund), andbody weight of a mouse was recorded as another measure of disease state.The mortality rate of the animals was recorded daily.

Although daily dosing of Copaxone™ was effective in reducing theseverity of the disease compared to mannitol dosing alone (FIG. 1),majority of the mice treated with daily dose of Copaxone™ died suddenlyafter about 3 weeks of treatment (FIG. 2). As shown in FIG. 3, dailydosing of Copaxone™ induced a large amount of antibodies in thesurviving injected mice. In contrast, weekly dosing with Copaxone™, anddaily and weekly dosing with Co-14 (YFAK), resulted in much lowerantibody titers. The immune response was predominantly IgG1+IgG2b (i.e.,predominantly TH2) responses, and a much lower IgG2a (i.e. TH1) responsewas seen. The few surviving mice in Copaxone™ daily group had large IgG1and IgG2b response against compound (FIGS. 4 and 5), raising thepossibility that the cause of death in Copaxone™ daily dosed mice islikely to be anaphylaxis. In contrast, weekly dosing with Copaxone™, anddaily and weekly dosing with Co-14 (YFAK), which showed a much lowerantibody titers, prevented anaphylactic shock and increased efficacy.Another example of antibody titers is shown in FIG. 6, where Copaxone™and Co-14 (YFAK) were administered either once a week or 3 times a week.Copaxone™, when administered 3 times a week, induces production of largeamount of antibodies directed against it, whereas weekly dosing ofCopaxone™ and dosing of Co-14, either weekly or three times a week, donot induce appreciative amount of antibodies against the respectivecopolymers.

When antibody titers for PLP (139-151) peptide were measured, bothCopaxone™ and Co-14 (YFAK), regardless of dosing interval, inducedsimilar, small increases in amounts of IgG1 formation against PLP(139-151) peptide (FIG. 7) compared to dosing with vehicle alone. Thetiters of IgG2b against PLP (139-151) were also not significantlyaffected (FIG. 8). These results show that the protective effect ofCopaxone™ or Co-14 (YFAK) is not exerted through modulation of antibodyamounts against PLP (139-151) peptide.

Example 2 T Cell Response to Random Copolymers

The TH1 and TH2 profiles of mice injected with 5 μg Copaxone™ or Co-14(YFAK) three times a week or on weekly bases, up to day 22 of thetreatment. On day 2, 8, 9, 15, 16, 22, 23, 29, spleens were collectedand splenocytes were isolated. 400,000 cells per well of splenocyteswere restimulated with various concentrations (0.8, 4, or 20 μg/ml) ofCo-14 (YFAK) for three days. On day 3 of the cell culture, the cellswere transferred onto ELISPOT (enzyme-linked immunospot assay) plates,coated with either IFN-γ (interferon gamma) or IL-13 (interleukin 13).The T cell response is examined by measuring the IFN γ production (a TH1cytokine) and IL-13 production (a TH2 cytokine). The degree of T cellstimulation is also examined by measuring the proliferation of the cellsshown as tritiated thymidine intake.

A burst of response was seen in the first week of dosing, followed by adecreased but sustained response. As seen in FIG. 9, the response is TH2biased, with the IL-13 production induced more strongly than the IFN-γat all times in cells treated with either Copaxone™ or Co-14 (YFAK). TheTH2 bias is further confirmed by the amount of 23 cytokines andchemokines, as seen in FIG. 10.

1. A method of reducing the severity of one or more symptoms of multiplesclerosis comprising the step of administering to a subject in needthereof an effective amount of a random copolymer composition, whereinsaid effective amount is delivered to said subject in two or more doses,each additional dose after the first dose being administered at leastone week after the conclusion of administration of the immediatelypreceding dose, and wherein the random copolymer composition comprisesYFAK (L-tyrosine, L-phenylalanine, L-alanine and L-lysine) in an outputmolar ratio of about 1.0:1.2:XA:6.0 respectively, and has a length of atleast 35 amino acids wherein XA=18.0 to 30.0.
 2. The method of claim 1,wherein the random copolymer composition comprises YFAK (L-tyrosine,L-phenylalanine, L-alanine and L-lysine) in an output molar ratio ofabout 1.0:1.2:XA:6.0 respectively, and has a length of at least 35 aminoacids wherein the composition is selected from the group consisting of:(a) a random copolymer composition comprising YFAK (L-tyrosine,L-phenylalanine, L-alanine and L-lysine) in an output molar ratio ofabout 1.0:1.2:20:6.0 respectively, and has a length of at least 35 aminoacids; and (b) a random copolymer composition comprising YFAK(L-tyrosine, L-phenylalanine, L-alanine and L-lysine) in an output molarratio of about 1.0:1.2:22:6.0 respectively, and has a length of at least35 amino acids; and (c) a random copolymer composition comprising YFAK(L-tyrosine, L-phenylalanine, L-alanine and L-lysine) in an output molarratio of about 1.0:1.2:24:6.0 respectively, and has a length of at least35 amino acids.
 3. The method of claim 1, wherein the multiple sclerosisis relapsing-remitting multiple sclerosis.
 4. The method of claim 1,wherein the subject is a human.
 5. The method of claim 1, wherein theeffective amount of the random copolymer is between 0.02 mg per dose and2000 mg per dose.
 6. The method of claim 1, wherein the effective amountis about 1 mg.
 7. The method of claim 1, wherein the random copolymercomposition is administered in a dosing regimen comprising a dose ofbetween 10 and 65 mg per m² of the body surface area of the subject perdose.
 8. The method of claim 7, wherein the dosing regimen comprises adose of about 22 mg per m² of the body surface area of the subject perdose.
 9. The method of claim 1, wherein the random copolymer compositionis administered in a dosing regimen comprising a dose of between about0.05 mg and 0.4 mg per kg of the body weight of the subject per dose.10. The method of claim 1, wherein the random copolymer composition isadministered in a dosing regimen comprising a maximum dose of about 140mg per m² of the body surface area of the subject weekly.
 11. The methodof claim 1, wherein the random copolymer composition is administered ina dosing regimen comprising a dose of between 0.35 and 2.8 mg per kg ofthe body weight of the subject weekly.
 12. The method of claim 1,wherein the dosing regimen comprises continuous administration of therandom copolymer composition.
 13. The method of claim 12, wherein theadministration of the random copolymer composition is via devicesdesigned to deliver the random copolymer composition continuously. 14.The method of claim 13, wherein the device is an implantable device, asustained release capsule, controlled release formulation, or a pump.15. The method of claim 12, wherein said effective amount is deliveredto the subject using a sustained-release formulation which administersthe random copolymer over a period of at least 3 days.
 16. The method ofclaim 1, further comprising administering an additional therapeuticallyactive agent to the subject.
 17. The method of claim 16, wherein theadditional agent is one or more random copolymers useful in treatingmultiple sclerosis.
 18. The method of claim 16, wherein the additionalagent is an anti-inflammatory agent.
 19. The method of claim 1, furthercomprising administering to said subject a T-cell depletion therapy. 20.The method of claim 1, further comprising administering to said subjectan anti-lymphocyte therapy.
 21. The method of claim 20, wherein saidanti-lymphocyte therapy comprises administering an agent selected fromthe group consisting of a polyclonal antibody and a monoclonal antibody.22. The method of claim 21, wherein said polyclonal antibody isantithymocyte gamma globulin (ATGAM).
 23. The method of claim 21,wherein said monoclonal antibody is selected from the group consistingof alemtuzumab, muromonab, daclizumab, and basiliximab.
 24. The methodof claim 1, further comprising administering to said subject an antiB-cell therapy.
 25. The method of claim 24, wherein said anti-B-celltherapy comprises administering anti CD-20 antibody.
 26. A method fordecreasing the frequency of recurrences of episodes of multiplesclerosis comprising the step of administering to a subject in needthereof an effective amount of a random copolymer composition, whereinsaid effective amount is delivered to said subject in two or more doses,each additional dose after the first dose being administered at leastone week after the administration of the immediately preceding dose, andwherein the random copolymer composition comprises YFAK (L-tyrosine,L-phenylalanine, L-alanine and L-lysine) in an output molar ratio ofabout 1.0:1.2:XA:6.0 respectively, and has a length of at least 35 aminoacids wherein XA=18.0 to 30.0.
 27. The method of claim 26, wherein therandom copolymer composition comprises YFAK (L-tyrosine,L-phenylalanine, L-alanine and L-lysine) in an output molar ratio ofabout 1.0:1.2:XA:6.0 respectively, and has a length of at least 35 aminoacids wherein the composition is selected from the group consisting of:(a) a random copolymer composition comprising YFAK (L-tyrosine,L-phenylalanine, L-alanine and L-lysine) in an output molar ratio ofabout 1.0:1.2:20:6.0 respectively, and has a length of at least 35 aminoacids; (b) a random copolymer composition comprising YFAK (L-tyrosine,L-phenylalanine, L-alanine and L-lysine) in an output molar ratio ofabout 1.0:1.2:22:6.0 respectively, and has a length of at least 35 aminoacids; and (c) a random copolymer composition comprising YFAK(L-tyrosine, L-phenylalanine, L-alanine and L-lysine) in an output molarratio of about 1.0:1.2:24:6.0 respectively, and has a length of at least35 amino acids.
 28. The method according to claim 1, wherein theeffective amount is delivered subcutaneously.
 29. The method accordingto claim 26, wherein the effective amount is delivered subcutaneously.